T cell epitopes of ryegrass pollen allergen

ABSTRACT

The present invention provides isolated peptides of Lol p V, a major protein allergen of the species  Lolium perenne . Therapeutic peptides within the scope of the invention comprise at least one T cell epitope, or preferably at least two T cell epitopes of a protein allergen of Lol p V. Diagnostic peptides within the scope of the invention bind IgE. The invention also provides modified peptides having similar or enhanced therapeutic properties or other desirable properties as the corresponding, naturally-occurring allergen or portion thereof. The invention further provides nucleic acid sequences coding for peptides of the invention. Use of the therapeutic compositions comprising one or more peptides of the invention in the manufacture of medicaments for treating sensitivity to Lol p V or an allergen immunologically related to Lol p V, or for general ryegrass sensitivity in an individual, is also provided. The invention also provides nucleic acid sequence coding for Dac g V protein allergen as well as the amino acid sequence of Dac g V protein allergen.

RELATED APPLICATIONS

This application is the National Stage of PCT/US94/09024, filed Aug. 5,1994 (published in English), which claims benefit of U.S. applicationSer. No. 08/106,016, filed Aug. 13, 1993 now abandoned.

BACKGROUND OF THE INVENTION

Allergens constitute the most abundant proteins of grass pollen, whichis the major cause of allergic disease in temperate climates (Marsh(1975) Allergens and the genetics of allergy; in M. Sela (ed.), TheAntigens, Vol. 3, pp 271–359, Academic Press Inc., London, N.Y.)., Hillet al. (1979) Medical Journal of Australia, 1:426–429). The firstdescriptions of the allergenic proteins in ryegrass showed that they areimmunochemically distinct, and are known as groups I, II, III and IV(Johnson and Marsh (1965) Nature, 206:935–942; and Johnson and Marsh(1966) Immunochemistry, 3:91–100). Using the International Union ofImmunological Societies' (IUIS) nomenclature, these allergens aredesignated Lol p I, Lol p II, Lol p III and Lol p IV. In addition,another important Lolium perenne L. allergen that has been identified inthe literature is Lol p IX which is also known as Lol p V or Lol p Ib(Singh et al. (1991) Proc. Natl. Acad. Sci, USA, 88:1384–1388).

These five proteins have been identified in pollen ryegrass, Loliumperenne L., and act as antigens in triggering immediate (Type 1)hypersensitivity in susceptible humans.

Lol p V is defined as an allergen because of its ability to bind tospecific IgE in sera of ryegrass-sensitive patients, to act as anantigen in IgG responses and to trigger T-cell responses. The allergenicproperties have been demonstrated by immunoblotting studies showing 80%of ryegrass pollen sensitive patients possessed specific IgE antibodythat bound to Lol p V isoforms (PCT application publication number WO93/04174, page 65). These results indicate that Lol p V is a majorryegrass allergen.

Substantial allergenic cross-reactivity between grass pollens has beendemonstrated using an IgE-binding assay, the radioallergo-sorbent test(RAST), for example, as described by Marsh et al. (1970) J. Allergy, 46,107–121, and Lowenstein (1978) Prog. Allergy, 25, 1–62. (Karger, Basel).

The immunochemical relationship of Lol p V with other grass pollenantigens have been demonstrated using both polyclonal and monoclonalantibodies (Zhang et al., Int. Arch Allergy Appl Immunol, 96:28–34(1991); Roberts et al., Int. Arch Allergy Appl Immunol, 98:178–180(1992); Mattheisen and Lowenstein, Clinical and Experimental Allergy,21:309–320 (1991); and van Ree et al., J. Allergy Clin. Immunol.83:144–151 (1989)). Antibodies have been prepared to purified proteinsthat bind IgE components. These data demonstrate that a major allergenis present in pollen of closely related grasses is immunochemicallysimilar to Lol p V and are generally characterized as Group V allergens.

In view of the prevalence of ryegrass pollen allergens and related grassallergens all over the world, there is a pressing need for thedevelopment of compositions and methods that could be used in detectingsensitivities to Lol p V or other immunologically related grassallergens, or in treating sensitivities to such allergens, or inassisting in the manufacture of medicaments to treat such sensitivities.The present invention provides materials and methods having one or moreof those utilities.

SUMMARY OF THE INVENTION

The present invention provides isolated peptides of Lol p V. Peptideswithin the scope of the invention comprise at least one T cell epitope,preferably at least two T cell epitopes of Lol p V. The inventionfurther provides peptides comprising at least two regions, each regioncomprising at least one T cell epitope of Lol p V.

The invention also provides modified peptides having similar or enhancedtherapeutic properties as the corresponding, naturally-occurringallergen or portion thereof, but having reduced side effects, as well asmodified peptides having improved properties such as increasedsolubility and stability. Therapeutic peptides of the invention arecapable of modifying, in a Lol p V-sensitive individual to whom they areadministered, the allergic response of the individual to Lol p V or anallergen immunologically cross-reactive Lol p V e.g. allergens derivedfrom pollen belonging to the Poacea (Graminae) family such as Dactylisglomerata, Dac g V.

Methods of treatment or of diagnosis of sensitivity to ryegrass pollenprotein, Lol p V in an individual or to pollen proteins that areimmunologically related to Lol p V such as Dac g V, and therapeuticcompositions comprising one or more peptides of the invention are alsoprovided.

The present invention also provides nucleic and amino acid sequences ofDac g V protein allergen which is immunologically cross-reative with Lolp V.

Further features of the present invention will be better understood fromthe following detailed description of the preferred embodiments of theinvention in conjunction with the appended figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A and FIG. 1B shows the nucleotide sequence of cDNA clone 12R (SEQID NO:1) and its predicted amino acid sequence (SEQ ID NO:2). Clone 12Ris a full-length clone of Lol p V derived from a λgtII library (see PCTapplication publication number WO93/04174).

FIG. 2 shows peptides of the invention of various lengths derived fromLol p V (SEQ ID NO: 3–29, 60).

FIG. 3 shows peptides of various lengths derived from Lol p I (SEQ IDNO:30–53, 55, 56, 61, 62).

FIG. 4 is a graphic representation depicting the response of T celllines from 19 patients primed in vitro with affinity purified Lol p Vand analyzed for response to Lol p V peptides (derived from the Lol p Vprotein allergen) by percent of responses with a mean S.I. of at least 2(indicated above each bar), the numbers enclosed in the parenthesisdenote percentage of patients responding to the particular peptide, andthe bar represents the positivity index for each peptide (% of patientsresponding multiplied by the mean S.I.).

FIG. 5 is a graphic representation derived from the same data shown inFIG. 4 showing the ranked sum for each peptide, the bar represents thecumulative rank of the peptide response in the group of 19 patientstested, above each bar in parenthesis is the percent of patientspositively responding to each peptide, the S.I. is also indicated aboveeach bar.

FIG. 6 is a graphic representation of the results of a direct ELISA, thesource of IgE was a sample of pooled human plasma (PHP) designatedPHP-A, and wherein the antigen is either soluble pollen extract (SPE) ofryegrass pollen, or bacterially expressed recombinant Lol p V (rLolpV).

FIG. 7 is a graphic representation of the results of a direct ELISA, thesource of IgE was a sample of pooled human plasma (PHP) designated PHP-Band wherein the antigen is either soluble pollen extract (SPE) ofryegrass pollen, rLol pV.

FIG. 8 is a graphic representation of the results of a direct ELISA, thesource of IgE was plasma from 4 individual patients, #1118, #1120,#1125, #1141, and wherein the antigen is ryegrass pollen SPE.

FIG. 9 is a graphic representation of the results of a direct ELISA thesource of IgE was plasma from 4 individual patients, #1118, #1120,#1125, #1141, and wherein the antigen is rLol p V.

FIG. 10 is a graphic representation of the results of a competitionELISA, the source of IgE was a sample of pooled human plasma designatedPHP-A, IgE binding was measured in the presence of ryegrass pollen SPE,affinity purified native Lol p V or rLol p V.

FIG. 11 is a graphic representation of the results of a competitionELISA, the source of IgE was plasma from individual patient #706 as asource of IgE, IgE binding was measured in the presence of ryegrasspollen SPE, affinity purified Lol p V or rLol p V.

FIG. 12 is a graphic representation of a histamine release assay toryegrass pollen SPE and rLol p V.

FIG. 13 a and FIG. 13 b each show a graphic representation of a directELISA using a sample of pooled human plasma designated PHP-B as a sourceof IgE, and wherein the antigen was either a selected peptide derivedfrom Lol p V or rLol p V.

FIG. 14 is a graphic representation of a competition ELISA using asample of pooled human plasma designated PHP-B as a source of IgE, andwherein the antigens were a mixture of affinity purified Lol p I and Lolp V or a mixture of recombinant Lol p I (rLol p I) or rLol p V tocompete for IgE binding to ryegrass pollen SPE.

FIG. 15 is a photograph of a Coomassie blue stained SDS-PAGE (12.5%)analysis of an Ab1B9-affinity purified native Lol p V, the sample wasrun under reducing conditions, the molecular weight standards are shownon the left.

FIG. 16A, FIG. 16B, and FIG. 16C show the nucleotide sequence of clone259 (SEQ ID NO:57) of Dac g V, and its predicted amino acid sequence(SEQ ID NO:58), the nucleotide sequence of nucleotides 1 to 699 has beenconfirmed, and the nucleotide sequence of nucleotides 700 to 1181 areunconfirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated peptides derived from Lol p V.The present invention also provides Dac g V protein allergen which isimmunologically cross-reactive with Lol p V. As used herein, a “peptide”refers to any protein fragment of Lol p V that induces an immuneresponse. The terms “fragment” and “antigenic fragment” as used hereinrefer to an amino acid sequence having fewer amino acid residues thanthe entire amino acid sequence of the protein from which the fragment isderived, and that induces an immune response. The terms “isolated” and“purified” as used herein refer to peptides of the invention which aresubstantially free of cellular material or culture medium when producedby recombinant DNA techniques, or substantially free of chemicalprecursors or other chemicals when synthesized chemically. As usedherein, the term “peptide” of the invention include peptides derivedfrom Lol p V which comprise at least one T cell epitope of the allergenor a portion of such peptide which comprises at least one T cellepitope.

Peptides comprising at least two regions, each region comprising atleast one T cell epitope of Lol p V are also within the scope of theinvention. Isolated peptides or regions of isolated peptides, eachcomprising at least two T cell epitopes of Lol p V protein allergen areparticularly desirable for increased therapeutic effectiveness. Peptideswhich are immunologically related (e.g., by antibody or T cellcross-reactivity) to peptides of the present invention, such as peptidesfrom Dac g V, are also within the scope of the invention. Peptidesimmunologically related by antibody cross-reactivity, are bound byantibodies specific for a peptide of Lol p V. Peptides immunologicallyrelated by T cell cross-reactivity are capable of reacting with the sameT cells as a peptide of the invention.

Isolated peptides of the invention can be produced by recombinant DNAtechniques in a host cell transformed with a nucleic acid having asequence encoding such peptide. The isolated peptides of the inventioncan also be produced by chemical synthesis. When a peptide is producedby recombinant techniques, host cells transformed with a nucleic acidhaving a sequence encoding a peptide of the invention or the functionalequivalent of the nucleic acid sequence are cultured in a mediumsuitable for the cells and peptides can be purified from cell culturemedium, host cells, or both using techniques known in the art forpurifying peptides and proteins including ion-exchange chromatography,gel filtration chromatography, ultrafiltration, electrophoresis orimmunopurification with antibodies specific for the peptide, the proteinallergen from which the peptide is derived, or a portion thereof.

The present invention provides expression vectors and host cellstransformed to express the nucleic acid sequences of the invention.Nucleic acid coding for a Lol p V peptide of the invention or at leastone fragment thereof may be expressed in bacterial cells such as E.coli, insect cells, yeast, or mammalian cells such as Chinese hamsterovary cells (CHO). Suitable expression vectors, promoters, enhancers,and other expression control elements may be found in Sambrook et al.Molecular Cloning: A Laboratory Manual, second edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Other suitableexpression vectors, promoters, enhancers, and other expression elementsare known to those skilled in the art. Suitable vectors for expressionin yeast include YepSec1 (Baldari et al. (1987) Embo J. 6: 229–234);pMFa (Kurjan and Herskowitz (1982) Cell 30: 933–943); JRY88 (Schultz etal. (1987) Gene 54: 113–123) and pYES2 (Invitrogen Corporation, SanDiego, Calif.). These vectors are freely available. Baculovirus andmammalian expression systems are also available. For example, abaculovirus system is commercially available (PharMingen, San Diego,Calif.) for expression in insect cells while the pMSG vector iscommercially available (Pharmacia, Piscataway, N.J.) for expression inmammalian cells.

For expression in E. coli, suitable expression vectors include, amongothers, pTRC (Amann et al. (1988) Gene 69: 301–315); pGEX (Amrad Corp.,Melbourne, Australia); pMAL (N.E. Biolabs, Beverly, Mass.); pRIT5(Pharmacia, Piscataway, N.J.); pET-11d (Novagen, Madison, Wis.) Jameelet al., (1990) J. Virol. 64:3963–3966; and pSEM (Knapp et al. (1990)BioTechniques 8: 280–281). The use of pTRC, and pET-11d, for example,will lead to the expression of unfused protein. The use of pMAL, pRIT5pSEM and pGEX will lead to the expression of allergen fused to maltose Ebinding protein (pMAL), protein A (pRIT5), truncated β-galactosidase(PSEM), or glutathione S-transferase (pGEX). When a Lol p V peptide ofthe invention is expressed as a fusion protein, it is particularlyadvantageous to introduce an enzymatic cleavage site at the fusionjunction between the carrier protein and Lol p V peptide. The Lol p Vpeptide may then be recovered from the fusion protein through enzymaticcleavage at the enzymatic site and biochemical purification usingconventional techniques for purification of proteins and peptides.Suitable enzymatic cleavage sites include those for blood clottingFactor Xa or thrombin for which the appropriate enzymes and protocolsfor cleavage are commercially available from, for example, SigmaChemical Company, St. Louis, Mo. and N.E. Biolabs, Beverly, Mass. Thedifferent vectors also have different promoter regions allowingconstitutive or inducible expression with, for example, IPTG induction(PRTC, Amann et al., (1988) supra; pET-11d, Novagen, Madison, Wis.) ortemperature induction (pRIT5, Pharmacia, Piscataway, N.J.). It may alsobe appropriate to express recombinant Lol p V peptides in different E.coli hosts that have an altered capacity to degrade recombinantlyexpressed proteins (e.g. U.S. Pat. No. 4,758,512). Alternatively, it maybe advantageous to alter the nucleic acid sequence to use codonspreferentially utilized by E. coli, where such nucleic acid alterationwould not affect the amino acid sequence of the expressed protein.

Host cells can be transformed to express the nucleic acid sequences ofthe invention using conventional techniques such as calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,or electroporation. Suitable methods for transforming the host cells maybe found in Sambrook et al. supra, and other laboratory textbooks. Thenucleic acid sequences of the invention may also be chemicallysynthesized using standard techniques (i.e. solid phase synthesis).Details of the isolation and cloning of clone 12R encoding Lol p V(described as Lol p Ib.1) are given in PCT application PublicationNumber WO 93/04174 incorporated herein by reference in its entirety.

Inducible non-fusion expression vectors include pTrc (Amann et al.,(1988) Gene, 69:301–315) and pET11d (Studier et al., Gene ExpressionTechnology: Methods in Enzymology, Academic Press, San Diego, Calif.(1990), 185:60–89). While target gene expression relies on host RNApolymerase transcription from the hybrid trp-lac fusion promoter inpTrc, expression of target genes inserted into pET11d relies ontranscription from the T7 gn10-lac 0 fusion promoter mediated bycoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21 (DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 under the transcriptional control of thelacUV 5 promoter.

One strategy to maximize recombinant Lol p V peptide expression in E.coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S., Gene Expression Technology: Methods in Enzymology, Academic Press,San Diego, Calif. (1990), 185:119–128). Another strategy would be toalter the nucleic acid sequence of the desired gene to be inserted intoan expression vector so that the individual codons for each amino acidwould be those preferentially utilized in highly expressed E. coliproteins (Wada et al. (1992) Nuc. Acids Res, 20:2111–2118). Suchalteration of nucleic acid sequences of the invention could be carriedout by standard DNA synthesis techniques.

The nucleic acids of the invention can also be chemically synthesizedusing standard techniques. Various methods of chemically synthesizingpolydeoxynucleotides are known, including solid-phase synthesis which,like peptide synthesis, has been fully automated in commerciallyavailable DNA synthesizers (See e.g., Itakura et al. U.S. Pat. No.4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S.Pat. Nos. 4,401,796 and 4,373,071, incorporated by reference herein).

The present invention also provides nucleic acid sequences encodingpeptides of the invention. Nucleic acid sequences used in any embodimentof this invention can be cDNAs encoding corresponding peptide sequencesas shown in FIG. 2 (SEQ ID NO:3–29, 60). Such oligodeoxynucleotidesequences can be produced chemically or mechanically, using knowntechniques. A functional equivalent of an oligonucleotide sequence isone which is 1) a sequence capable of hybridizing to a complementaryoligonucleotide to which the sequence (or corresponding sequenceportions) of Lol p V as shown in FIG. 1 or fragments thereof hybridizes,or 2) the sequence (the corresponding sequence portions complementary tothe nucleic acid sequences encoding the peptide sequence derived fromLol p V as shown in FIGS. 2 and/or 3) a sequence which encodes a product(e.g., a polypeptide or peptide) having the same functionalcharacteristics of the product encoded by the sequence (or correspondingsequence portion) of Lol p V as shown in FIG. 1. Whether a functionalequivalent must meet one or more criteria will depend on its use (e.g.,if it is to be used only as an oligoprobe, it need meet only the firstor second criteria and if it is to be used to produce a Lol p V peptideof the invention, it need only meet the third criterion). The nucleicacid sequences of the invention also include RNA which can betranscribed from the DNA prepared as described above.

Preferred nucleic acids encode a peptide having at least about 50%homology to a Lol p V peptide of the invention, more preferably at leastabout 60% homology and most preferably at least about 70% homology witha Lol p V peptide of the invention. Nucleic acids that encode peptideshaving at least about 90%, more preferably at least about 95%, and mostpreferably at least about 98–99% homology with Lol p V peptides of theinvention are also within the scope of the invention. Homology refers tosequence similarity between two peptides of Lol p V, or between twonucleic acid molecules. Homology can be determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame nucleotide or amino acid, then molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences.

Preferred nucleic acid fragments encode peptides of at least 7 aminoacid residues in length, and preferably 13–40 amino acid residues inlength, and more preferably at least 16–30 amino acids residues inlength, Nucleic acid fragments encoding peptides of at least 30 aminoacid residues in length, at least 40 amino acid residues in length, atleast about 80 amino acid residues in length, at least about 100 aminoacid residues in length or more, are also contemplated.

Also within the scope of the invention are nucleic acid sequencesencoding allergens immunologically cross-reactive with Lol p V, such asfull length Dac g V protein or peptides (FIG. 16). Proteins and peptidesof Dac g V may be produced recombinantly as discussed above, orsynthetically. Expression vectors and host cells transformed to expressDac g V protein or peptides thereof are also within the scope of theinvention. Details of the cloning of Dac g V are given in the examples.

The present invention also provides a method of producing isolated Lol pV peptides of the invention or a portion thereof comprising the steps ofculturing a host cell transformed with a nucleic acid sequence encodinga Lol p V peptide of the invention in an appropriate medium to produce amixture of cells and medium containing said Lol p V peptide; andpurifying the mixture to produce substantially pure Lol p V peptide.Host cells transformed with an expression vector containing DNA codingfor a Lol p V peptide of the invention or a portion thereof are culturedin a suitable medium for the host cell. Lol p V peptides of theinvention can be purified from cell culture medium, host cells, or bothusing techniques known in the art for purifying peptides and proteinsincluding ion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis and immunopurification with antibodiesspecific for the Lol p V peptides or portions thereof of the invention.

Another aspect of the present invention pertains to an antibodyspecifically reactive with a Lol p V peptide. Such antibodies may beused to standardize allergen extracts or to isolate the naturallyoccurring Lol p V. Also, Lol p V peptides of the invention can be usedas “purified” allergens to standardize allergen extracts. For example,an animal such as a mouse or rabbit can be immunized with an immunogenicform of an isolated Lol p V peptide of the invention capable ofeliciting an antibody response. Techniques for conferring immunogenicityon a peptide include conjugation to carriers or other techniqueswell-known in the art. The Lol p V peptide also can be administered inthe presence of adjuvant. The progress of immunization can be monitoredby detection of antibody titers in plasma or serum standard ELISA orother immunoassay can be used with the immunogen as antigen to assessthe levels of antibodies.

Following immunization, anti-Lol p V peptide antisera can be obtainedand, if desired, polyclonal anti-Lol p V peptide antibodies from theserum. To produce monoclonal antibodies, antibody producing cells(lymphocytes) can be harvested from an immunized animal and fused bystandard somatic cell fusion procedures with immortalizing cells such asmyeloma cells to yield hybridoma cells. Hybridoma cells can be screenedimmunochemically for production of antibodies reactive with the Lol p Vpeptides of the invention. These sera or monoclonal antibodies can beused to standardize allergen extracts.

Through use of the peptides and antibodies of the present invention,preparations of consistent, well-defined composition and biologicalactivity can be made and administered for therapeutic purposes (e.g. tomodify the allergic response of a ryegrass pollen sensitive individualto pollen of such grasses or pollen of an immunologically related grasssuch as Dac g V). Administration of such peptides may, for example,modify B-cell response to Lol p V allergen, T-cell response to Lol p Vallergen or both responses. Isolated peptides can also be used to studythe mechanism of immunotherapy of ryegrass pollen allergy and to designmodified derivatives or analogues useful in immunotherapy.

The present invention also pertains to T cell clones which specificallyrecognize Lol p V peptides of the invention. These T cell clones may besuitable for isolation and molecular cloning of the gene for the T cellreceptor which is specifically reactive with a peptide of the presentinvention. The T cell clones may be produced as described in Cellularand Molecular Immunology, Abdul K. Abbas et al., W.B. Saunders Co.(1991) pg. 139. The present invention also pertains to soluble T cellreceptors. These receptors may inhibit antigen-dependent activation ofthe relevant T cell subpopulation within an individual sensitive to Lolp V. Antibodies specifically reactive with such a T cell receptor canalso be produced according to the techniques described herein. Suchantibodies may also be useful to block T-cell -MHC interaction in anindividual. Methods for producing soluble T cell receptors are describedin Immunology; A Synthesis, 2nd Ed., Edward S. Golub et al., SinaurAssoc, Sunderland Mass., (1991) pp. 366–369.

To obtain isolated peptides of the present invention, Lol p V is dividedinto non-overlapping peptides of desired length or overlapping peptidesof desired lengths as discussed in Example 2 which can be producedrecombinantly, synthetically, or in certain situations, by chemicalcleavage of the allergen. Peptides comprising at least one T cellepitope are capable of eliciting a T cell response, such as stimulation(i.e. proliferation or lymphokine secretion) and/or are capable ofinducing T cell non-responsiveness. To determine peptides comprising atleast one T cell epitope, isolated peptides are tested by, for example,T cell biology techniques, to determine whether the peptides elicit a Tcell response or induce T cell non-responsiveness. Those peptides foundto elicit a T cell response or induce T cell non-responsiveness aredefined as having T cell stimulating activity.

Screening peptides of the invention for human T cell stimulatingacitivity can be accomplished using one or more of several differentassays. For example, in vitro, T cell stimulatory activity is assayed bycontacting a peptide of the invention with an antigen presenting cellwhich presents appropriate MHC molecules in a T cell culture.Presentation of a peptide of the invention in association withappropriate MHC molecules to T cells, in conjunction with the necessarycostimulation has the effect of transmitting a signal to the T cell thatinduces the production of increased levels of cytokines, particularly ofinterleukin-2 and interleukin-4. The culture supernatant can be obtainedand assayed for interleukin-2 or other known cytokines. For example, anyone of several conventional assays for interleukin-2 can be employed,such as the assay described in Proc. Natl. Acad. Sci USA, 86:1333 (1989)the pertinent portions of which are incorporated herein by reference. Akit for an assay for the production of interferon is also available fromGenzyme Corporation (Cambridge, Mass.).

A common assay for T cell proliferation entails measuring tritiatedthymidine incorporation. The proliferation of T cells can be measured invitro by determining the amount of ³H-labeled thymidine incorporatedinto the replicating DNA of cultured cells. Therefore, the rate of DNAsynthesis and, in turn, the rate of cell division can be quantified.

A peptide may also be screened for the ability to reduce T cellresponsiveness. The ability of a peptide known to stimulate T cells, toinhibit or completely block the activity of a purified native Lol p Vprotein allergen or portion thereof and induce a state of T cellnonresponsiveness or reduced T cell responsiveness, can be determinedusing subsequent attempts at stimulation of the T cells with antigenpresenting cells that present a native Lol p V allergen followingexposure to a peptide of the invention. If the T cells are unresponsiveto the subsequent activation attempts, as determined by interleukin-2synthesis and T cell proliferation, a state of nonresponsiveness hasbeen induced. See, e.g., Gimmi, et al. (1993) Proc. Natl. Acad. Sci USA,90:6586–6590; and Schwartz (1990) Science, 248:1349–1356, for assaysystems that can be used as the basis for an assay in accordance withthe present invention.

Additionally, peptides comprising “cryptic epitopes” may be determinedand are also within the scope of this invention. Cryptic epitopes arethose determinants in a protein antigen which, due to processing andpresentation of the native protein antigen to the appropriate MHCmolecule, are not normally revealed to the immune system. However, apeptide comprising a cryptic epitope is capable of causing T cells tobecome non-responsive, and when a subject is primed with the peptide, Tcells obtained from the subject will proliferate in vitro in response tothe peptide or the protein antigen from which the peptide is derived.Peptides which comprise at least one cryptic epitope derived from aprotein antigen are referred to herein as “cryptic peptides”. To confirmthe presence of cryptic epitopes in the above-described T cellproliferation assay, antigen-primed T cells are cultured in vitro in thepresence of each peptide separately to establish peptide-reactive T celllines. A peptide is considered to comprise at least one cryptic epitopeif a T cell line can be established with a given peptide and T cells arecapable of proliferation upon challenge with the peptide and the proteinantigen from which the peptide is derived.

It is also possible to modify the structure of a peptide of theinvention for such purposes as increasing solubility, enhancingtherapeutic or preventive efficacy, or stability (e.g., shelf life exvivo, and resistance to proteolytic degradation in vivo). A modifiedpeptide can be produced in which the amino acid sequence has beenaltered, such as by amino acid substitution, deletion, or addition, tomodify immunogenicity and/or reduce allergenicity, or to which acomponent has been added for the same purpose.

For example, a peptide can be modified so that it maintains the abilityto induce T cell anergy and bind MHC proteins without the ability toinduce a strong proliferative response or possibly, any proliferativeresponse when administered in immunogenic form. In this instance,critical binding residues for the T cell receptor can be determinedusing known techniques (e.g., substitution of each residue anddetermination of the presence or absence of T cell reactivity). Thoseresidues shown to be essential to interact with the T cell receptor canbe modified by replacing the essential amino acid with another,preferably similar amino acid residue (a conservative substitution)whose presence is shown to enhance, diminish but not eliminate, or notaffect T cell reactivity. In addition, those amino acid residues whichare not essential for T cell receptor interaction can be modified bybeing replaced by another amino acid whose incorporation may enhance,diminish or not affect T cell reactivity but does not eliminate bindingto relevant MHC.

Additionally, peptides of the invention can be modified by replacing anamino acid shown to be essential to interact with the MHC proteincomplex with another, preferably similar amino acid residue(conservative substitution) whose presence is shown to enhance, diminishbut not eliminate, or not affect T cell activity. In addition, aminoacid residues which are not essential for interaction with the MHCprotein complex but which still bind the MHC protein complex can bemodified by being replaced by another amino acid whose incorporation mayenhance, not affect, or diminish but not eliminate T cell reactivity.Preferred amino acid substitutions for non-essential amino acidsinclude, but are not limited to substitutions with alanine, glutamicacid, or a methyl amino acid.

In order to enhance stability and/or reactivity, peptides of theinvention can also be modified to incorporate one or more polymorphismsin the amino acid sequence of the protein allergen resulting fromnatural allelic variation. Additionally, D-amino acids, non-naturalamino acids or non-amino acid analogues can be substituted or added toproduce a modified peptide within the scope of this invention.Furthermore, peptides of the present invention can be modified using thepolyethylene glycol (PEG) method of A. Sehon and co-workers (Wie et al.supra) to produce a protein or peptide conjugated with PEG. In addition,PEG can be added during chemical synthesis of a protein or peptide ofthe invention. Modifications of peptides or portions thereof can alsoinclude reduction/alkylation (Tarr in: Methods of ProteinMicrocharacterization, J. E. Silver ed. Humana Press, Clifton, N.J., pp155–194 (1986)); acylation (Tarr, supra); chemical coupling to anappropriate carrier (Mishell and Shiigi, eds., Selected Methods inCellular Immunology, WH Freeman, San Francisco, Calif. (1980); U.S. Pat.No. 4,939,239; or mild formalin treatment (Marsh, International Archivesof Allergy and Applied Immunology, 41:199–215 (1971)).

To facilitate purification and potentially increase solubility ofpeptides of the invention, it is possible to add reporter group(s) tothe peptide backbone. For example, poly-histidine can be added to apeptide to purify the peptide on immobilized metal ion affinitychromatography (Hochuli, E. et al., Bio/Technology, 6:1321–1325 (1988)).In addition, specific endoprotease cleavage sites can be introduced, ifdesired, between a reporter group and amino acid sequences of a peptideto facilitate isolation of peptides free of irrelevant sequences. Inorder to successfully desensitize an individual to a protein antigen, itmay be necessary to increase the solubility of a peptide by addingfunctional groups to the peptide or by not including hydrophobic T cellepitopes or regions containing hydrophobic epitopes in the peptides orhydrophobic regions of the protein or peptide. Functional groups such ascharged amino acid pairs (e.g., KK or RR) are particularly useful forincreasing the solubility of a peptide when added to the amino orcarboxy terminus of the peptide.

To potentially aid proper antigen processing of T cell epitopes within apeptide, canonical protease sensitive sites can be recombinantly orsynthetically engineered between regions, each comprising at least one Tcell epitope. For example, charged amino acid pairs, such as KK or RR,can be introduced between regions within a peptide during recombinantconstruction of the peptide. The resulting peptide can be renderedsensitive to cathepsin and/or other trypsin-like enzymes cleavage togenerate portions of the peptide containing one or more T cell epitopes.In addition, as discussed above, such charged amino acid residues can beadded to the amino or carboxy terminus of the peptide and can result inan increase in solubility of a peptide.

Site-directed mutagenesis of DNA encoding a peptide of the invention canbe used to modify the structure of the peptide by methods known in theart. Such methods may, among others, include PCR with oligonucleotidescontaining the sequences encoding the desired amino acids (Ho et al.,Gene, 77:51–59 (1989)) or total synthesis of mutated genes (Hostomsky,Z. et al., Biochem. Biophys, Res. Comm., 161:1056–1063 (1989)). Toenhance bacterial expression, the aforementioned methods can be used inconjunction with other procedures to change the eukaryotic codons in DNAconstructs encoding protein or peptides of the invention to onespreferentially used in E. coli, yeast, mammalian cells, or othereukaryotic cells.

Peptides or antibodies of the present invention can also be used fordetecting and diagnosing ryegrass pollinosis. For example, this could bedone in vitro by combining blood or blood products obtained from anindividual to be assessed for sensitivity to ryegrass pollen or anothercross reactive pollen such as Dac g V, with isolated peptides of Lol pV, under conditions appropriate for binding of components in the blood(e.g., antibodies, T cells, B cells) with the peptide(s) and determiningthe extent to which such binding occurs. Other diagnostic methods forallergic diseases in which the protein, peptides or antibodies of thepresent invention will be useful include radio-allergergosorbent test(RAST), paper radioimmunosorbent test (PRIST), enzyme linkedimmunosorbent assay (ELISA), radioimmunoassays (RIA), immuno-radiometricassays (IRMA), luminescence immunoassays (LIA), histamine release assaysand IgE immunoblots.

The presence in individuals of IgE specific for at least one proteinallergen and the ability of T cells of the individuals to respond to Tcell epitope(s) of the protein allergen can be determined byadministering to the individuals an Immediate Type Hypersensitivity testand a Delayed Type Hypersensitiity test. The individuals areadministered an Immediate Type Hypersensitivity test (see e.g.,Immunology (1985) Roitt, I. M., Brostoff, J., Male, D. K. (eds), C.V.Mosby Co., Gower Medical Publishing, London, N.Y., pp. 19.2–19.18; pp.22.1–22.10) utilizing the protein allergen or a portion thereof, or amodified form of the protein allergen or a portion thereof, each ofwhich binds IgE specific for the allergen. The same individuals areadministered a Delayed Type Hypersensitivity test prior to,simultaneously with, or subsequent to administration of the ImmediateType Hypersensitivity test. Of course, if the Immediate TypeHypersensitivity test is administered prior to the Delayed TypeHypersensitivity test, the Delayed Type Hypersensitivity test would begiven to those individuals exhibiting a specific Immediate TypeHypersensitivity reaction. The Delayed Type Hypersensitivity testutilizes a modified form of the protein allergen or a portion thereof,the protein allergen produced recombinantly, or a peptide derived fromthe protein allergen, each of which has human T cell stimulatingactivity and each of which does not bind IgE specific for the allergenin a substantial percentage of the population of individuals sensitiveto the allergen (e.g., at least about 75%). Those individuals found tohave both a specific Immediate Type Hypersensitivity reaction and aspecific Delayed Type Hypersensitivity reaction may be treated with atherapeutic composition comprising the same modified form of the proteinor portion thereof, the recombinantly produced protein allergen, or thepeptide, each as used in the Delayed Type Hypersensitivity test.

Isolated peptides of the invention when administered in a therapeuticregimen to a Lol p V-sensitive individual, or an individual allergic toan allergen cross-reactive with Lol p V such as Dac g V, are capable ofmodifying the allergic response of the individual to Lol p V ryegrasspollen allergen or such cross-reactive allergen, and preferably arecapable of modifying the B-cell response, T-cell response or both theB-cell and the T-cell response of the individual to the allergen. Asused herein, modification of the allergic response of an individualsensitive to a ryegrass pollen allergen or cross-reactive allergen canbe defined as non-responsiveness or diminution in symptoms to theallergen, as determined by standard clinical procedures (See e.g. Varneyet al, British Medical Journal, 302:265–269 (1990)) including diminutionin ryegrass pollen induced asthmatic symptoms. As referred to herein, adiminution in symptoms includes any reduction in the allergic responseof an individual to the allergen after the individual has completed atreatment regimen with a peptide or protein of the invention. Thisdiminution may be subjective (i.e. the patient feels more comfortable inthe presence of the allergen). Diminution in symptoms can be determinedclinically as well, using standard skin tests as is known in the art.

Lol p V peptides of the present invention which have T cell stimulatingactivity, and thus comprise at least one T cell epitope are particularlydesirable for therapeutic purposes. In referring to an epitope, theepitope will be the basic element or smallest unit of recognition by areceptor, particularly immunoglobulins, histocompatibility antigens andT cell receptors where the epitope comprises amino acids essential toreceptor recognition. Amino acid sequences which mimic those of theepitopes and which are capable of down regulating or reducing allergicresponse to Lol p V can also be used. T cell epitopes are believed to beinvolved in initiation and perpetuation of the immune response to aprotein allergen which is responsible for the clinical symptoms ofallergy. These T cell epitopes are thought to trigger early events atthe level of the T helper cell by binding to an appropriate HLA moleculeon the surface of an antigen presenting cell and stimulating therelevant T cell subpopulation. These events lead to T cellproliferation, lymphokine secretion, local inflammatory reactions,recruitment of additional immune cells to the site, and activation ofthe B cell cascade leading to production of antibodies. One isotype ofthese antibodies, IgE, is fundamentally important to the development ofallergic symptoms and its production is influenced early in the cascadeof events, at the level of the T helper cell, by the nature of thelymphokines secreted.

Exposure of ryegrass pollen patients to isolated Lol p V peptides of thepresent invention which comprise at least one T cell epitope and arederived from Lol p V protein allergen may cause appropriate T cellsubpopulations to become nonresponsive or have a reduced response to theprotein allergen and thus do not participate in stimulating an immuneresponse upon such exposure. In addition, administration of a peptide ofthe invention or portion thereof which comprises at least one T cellepitope may modify the lymphokine secretion profile as compared withexposure to the naturally-occurring Lol p V protein allergen or portionthereof (e.g. result in a decrease of IL-4 and/or an increase in IL-2).Furthermore, administration of such peptide of the invention mayinfluence T cell subpopulations which normally participate in theresponse to the naturally occurring allergen such that these T cells aredrawn away from the site(s) of normal exposure to the allergen (e.g.,nasal mucosa, skin, and lung) towards the site(s) of therapeuticadministration of the fragment or protein allergen. This redistributionof T cell subpopulations may ameliorate or reduce the ability of anindividual's immune system to stimulate the usual immune response at thesite of normal exposure to the allergen, resulting in a diminution inallergic symptoms.

The isolated Lol p V peptides of the invention can be used in methods ofdiagnosing, treating and preventing allergic reactions to Lol p Vallergen or a cross reactive protein allergen. Thus the presentinvention provides compositions useful in allergy diagnosis and/oruseful in allergy therapy comprising isolated Lol p V peptides orportions thereof. Such compositions will typically also comprise apharmaceutically acceptable carrier or diluent when intended for in vivoadministration. Therapeutic compositions of the invention may alsocomprise synthetically prepared Lol p V peptides and a pharmaceuticallyacceptable carrier or diluent.

Administration of the therapeutic compositions of the present inventionto an individual to be desensitized can be carried out using knowntechniques. Lol p V peptides or portions thereof may be administered toan individual in combination with, for example, an appropriate diluent,a carrier and/or an adjuvant. Pharmaceutically acceptable diluentsinclude saline and aqueous buffer solutions. Pharmaceutically acceptablecarriers include polyethylene glycol (Wie et al. (1981) Int. ArchAllergy Appl. Immunol. 64:84–99) and liposomes (Strejan et al. (1984) J.Neuroimmunol, 7: 27).

The therapeutic compositions of the invention are administered toryegrass allergen sensitive individuals or individuals sensitive to anallergen which is immunologically cross-reactive with house ryegrassallergen (i.e. Dactylis glomerata, or Sorghum halepensis, etc.). For:

the purposes of inducing T cell non responsiveness, therapeuticcompositions of the invention are preferably administered innon-immunogenic form, e.g. which does not contain adjuvant. While notintending to be limited to any theory, it is believed that T cell nonresponsiveness or reduced T cell responsiveness is induced as a resultof not providing an appropriate costimulatory signal sometimes referredto as a “second signal” Briefly, it is believed that stimulation of Tcells requires two types of signals, the first is the recognition by theT cell via the T cell receptor of appropriate MHC-associated processedantigens on antigen presenting cells (APCs) and the second type ofsignal is referred to as a costimulatory signal(s) or “second signal”which may be provided by certain competent APCs. When a composition ofthe invention is administered without adjuvant, it is believed thatcompetent APCs which are capable of producing the second signal orcostimulatory signal are not engaged in the stimulation of appropriate Tcells therefore resulting in T cell nonresponsiveness or reduced T cellresponsiveness. In addition, there are a number of antibodies or otherreagents capable of blocking the delivery of costimulatory signals suchas the “second signal” which include, but are not limited to B7(including B7-1, B7-2, and BB-1), CD28, CTLA4, CD40 CD40L CD54 andCD11a/18 (Jenkins and Johnson, Current Opinion in Immunology, 5:361–367(1993), and Clark and Ledbetter, Nature, 367:425–428 (1994)) Thus, apeptide of the invention may be administered in nonimmunogenic form asdiscussed above, in conjunction with a reagent capable of blockingcostimulatory signals such that the level of T cell nonresponsiveness isenhanced.

Administration of the therapeutic compositions of the present inventionto an individual to be desensitized can be carried out using knownprocedures at dosages and for periods of time effective to reducesensitivity (i.e., reduce the allergic response) of the individual tothe allergen. Effective amounts of the therapeutic compositions willvary according to factors such as the degree of sensitivity of theindividual to ryegrass pollen, the age, sex, and weight of theindividual, and the ability of the protein or fragment thereof to elicitan antigenic response in the individual.

The active compound (i.e., protein or fragment thereof) may beadministered in a convenient manner such as by injection (subcutaneous,intravenous, etc.), oral administration, inhalation, transdermalapplication, or rectal administration. Depending on the route ofadministration, the active compound may be coated within a material toprotect the compound from the action of enzymes, acids and other naturalconditions which may inactivate the compound.

For example, preferably about 1 μg–3 mg and more preferably from about20–750 μg of active compound (i.e., protein or fragment thereof) perdosage unit may be administered by injection. Dosage regimen may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation.

To administer a peptide by other than parenteral administration, it maybe necessary to coat the protein with, or co-administer the proteinwith, a material to prevent its inactivation. For example, peptide orportion thereof may be co-administered with enzyme inhibitors or inliposomes. Enzyme inhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEP) and trasylol. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al., (1984) J. Neuroimmunol., 7:27).

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethyline glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions of dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glyceral,propylene glycol, and liquid polyetheylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thirmerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmanitol and sorbitol or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about,including in the composition, an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating activecompound (i.e., protein or peptide) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile indectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient (i.e., protein orpeptide) plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

When a peptide of the invention is suitably protected, as describedabove, the peptide may be orally administered, for example, with aninert diluent or an assimilable edible carrier. The peptide and otheringredients may also be enclosed in a hard or soft shell gelatincapsule, compressed into tablets, or incorporated directly into theindividual's diet. For oral therapeutic administration, the activecompound may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the composition and preparations may, of course, bevaried and may conveniently be between about 5 to 80% of the weight ofthe unit. The amount of active compound in such therapeutically usefulcompositions is such that a suitable dosage will be obtained. Preferredcompositions or preparations according to the present invention areprepared so that an oral dosage unit contains between from about 10 μgto about 200 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup or elixir may contain the active compound, sucrose as a sweeteningagent, methyl and propylparabens as preservative, a dye and flavoringsuch as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompound may be incorporated into sustained-release preparations andformulations.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the therapeuticcompositions is contemplate Supplementary active compounds can also beincorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit from as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

Various isolated peptides of the invention derived from ryegrass pollenprotein Lol p V are shown in FIG. 2 (SEQ ID NO:3–29, 60). Peptidescomprising at least two regions, each region comprising at least one Tcell epitope of Lol p V are also within the scope of the invention. Asused herein a region may include the amino acid sequence of a peptide ofthe invention as shown in FIG. 2 or the amino acid sequence of a portionof such peptide.

As discussed in Example 2, human T cell stimulating activity can betested by culturing T cells obtained from an individual sensitive to Lolp V allergen, (i.e., an individual who has an IgE mediated immuneresponse to Lol p V allergen) with a peptide derived from the allergenand determining whether proliferation of T cells occurs in response tothe peptide as measured, e.g., by cellular uptake of tritiatedthymidine. Stimulation indices for responses by T cells to peptides canbe calculated as the maximum CPM in response to a peptide divided by thecontrol CPM. A stimulation index (S.I.) equal to or greater than twotimes the background level is considered “positive”. Positive resultsare used to calculate the mean stimulation index for each peptide forthe group of patients tested. In FIGS. 4 and 5 the mean T cellstimulation index is indicated above the bar. Preferred peptides of thisinvention comprise at least one T cell epitope and have a mean T cellstimulation index of greater than or equal to 2.0. A peptide having amean T cell stimulation index of greater than or equal to 2.0 in asignificant number of ryegrass pollen sensitive patients tested isconsidered useful as a therapeutic agent. Preferred peptides have a meanT cell stimulation index of at least 2.5, more preferably at least 3.0,more preferably at least 3.5, more preferably at least 4.0, morepreferably at least 5.0 and most preferably at least about 6. Forexample, peptides of the invention having a mean T cell stimulationindex of at least 5, as indicated by data shown in FIGS. 4 and 5,include peptides LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-8 (SEQID NO:10), LPIX-17 (SEQ ID NO:19) and LPIX-19 (SEQ ID NO:21).

In addition, preferred peptides have a positivity index (P.I.) of atleast about 60, more preferably about 100, more preferably at leastabout 200 and most preferably at least about 300. The positivity indexfor a peptide is determined by multiplying the mean T cell stimulationindex by the percent of individuals, in a population of individualssensitive to ryegrass pollen (e.g., preferably a population of at least15 individuals, more preferably a population of at least 30 individualsor more), who have a T cell stimulation index to such peptide of atleast 2.0. Thus, the positivity index represents both the strength of aT cell response to a peptide (S.I.) and the frequency of a T cellresponse to a peptide in a population of individuals sensitive toryegrass pollen. In FIG. 4, the bar represents the positivity index andthe percent of individuals tested who have a T cell stimulation index ofat least 2.0 to that peptide are indicated in parenthesis above each bar(the mean T cell stimulation index is also indicated above each bar).For example, as shown in FIG. 4, Lol p V peptide LPIX-5 (SEQ ID NO:7)has a mean S.I. of 5.8 and 26.3% of positive responses in the group ofindividuals tested resulting in a positivity index of 152.54. Lol p Vpeptides having a positivity index of at least about 100 and a mean Tcell stimulation index of at least about 4 include: LPIX-4 (SEQ IDNO:6), LPIX-5 (SEQ ID NO:7), and LPIX-17 (SEQ ID NO:19).

In FIG. 5, the bar represents the cumulative rank of the peptideresponse in the group of patients tested as described in Example 2. Todetermine the cumulative rank, the 5 peptides with the highest S.I. ineach individual were determined and assigned a numerical rank indescending order, with 5 representing the strongest response. The ranksfor each peptide were then summed for the entire group of patientstested to determine the cumulative rank for the peptide. Above each baris the mean S.I. for each peptide and the percent of positive responses(in parenthesis) with an S.I. of at least 2 to the peptide in the groupof patients tested.

In order to determine precise T cell epitopes by, for example, finemapping techniques, a peptide having T cell stimulating activity andthus comprising at least one T cell epitope as determined by T cellbiology techniques is modified by addition or deletion of amino acidresidues at either the amino or carboxy terminus of the peptide andtested to determine a change in T cell reactivity to the modifiedpeptide. Following this technique, peptides are selected and producedrecombinantly or synthetically. Peptides are selected based on variousfactors, including the strength of the T cell response to the peptide(e.g., stimulation index), the frequency of the T cell response to thepeptide in a population of individuals sensitive to ryegrass pollen, andthe potential cross-reactivity of the peptide with other allergens fromother species of grasses as discussed earlier i.e. Dactylis glomerata.The physical and chemical properties of these selected peptides (e.g.,solubility, stability) are examined to determine whether the peptidesare suitable for use in therapeutic compositions or whether the peptidesrequire modification as described herein. The ability of the selectedpeptides or selected modified peptides to stimulate human T cells (e.g.,induce proliferation, lymphokine secretion) or cause appropriate T cellpopulations to become non-responsive or have a reduced response to theprotein allergen is determined.

In addition, it may be desirable to further modify peptides such asLPIX-4 (SEQ ID NO:6), -5 (SEQ ID NO:7), -6 (SEQ ID NO:8), -11 (SEQ IDNO:13), -12 (SEQ ID NO:14), -16 (SEQ ID NO:18), -17 (SEQ ID NO:19) and-20 (SEQ ID NO:22) for purposes of increasing solubility or stability.Modifications to improve solubility include truncation from either theamino or carboxyl terminus of the peptide or both termini to removehydrophilic amino acids such as Val, Ile, Leu, Phe, Tyr and Trp.Residues removed by truncation may also be replaced with chargedhydrophilic amino acids such as Asp, Glu, Lys and Arg or neutralhydrophilic amino acids such as Ser, Pro, Gly or Ala. Such amino acidsmay be of either the R or S optical configuration.

Other modifications to improve solubility include attachment ofhydrophilic polymers to either the amino- or carboxy terminus of thepeptides or to both. Such polymers may be polyanionic, polycationic orneutral (such as polyoxyethylene).

Modifications to improve stability include deletion or replacement ofAsn and Gln residues and elimination ot Asn-Gly, Asp-Gly and Asp-Prosequences.

Specific examples of modifications listed above would be removal of theN-terminal Val and C-terminal Val-His-Ala-Val from peptide LIX-12. Theresulting truncated peptide could be used directly or the deletedresidues could be replaced by combinations of the polar amino acids Asp,Glu, Lys and Arg. Similarly, the N-terminal sequence Gly-Phe andC-terminal sequence Phe-Lys-Ile could be removed from peptide LPIX-5(SEQ ID NO:7).

Additionally, preferred T cell epitope-containing peptides of theinvention do not bind immunoglobulin E (IgE) or bind IgE to asubstantially lesser extent (e.g. at least 100 fold less and morepreferably at least 1000 fold less) than the protein allergen from whichthe peptide is derived. The major complications of standardimmunotherapy are IgE-mediated responses such as anaphylaxis.Immunoglobulin E is a mediator of anaphylactic reactions which resultfrom the binding and cross-linking of antigen to IgE on mast cells orbasophils and the release of mediators (e.g., histamine, serotonin,eosinophil chemotacic factors). Thus, anaphylaxis in a substantialpercentage of a population of individuals sensitive to Lol p V could beavoided by the use in immunotherapy of a peptide or peptides which donot bind IgE in a substantial percentage (e.g., at least about 75%) of apopulation of individuals sensitive to Lol p V allergen, or if thepeptide binds IgE, such binding does not result in the release ofmediators from mast cells or basophils. The risk of anaphylaxis could bereduced by the use in immunotherapy of a peptide or peptides which havereduced IgE binding. Moreover, peptides which have minimal IgEstimulating activity are desirable for therapeutic effectiveness.Minimal IgE stimulating activity refers to IgE production that is lessthan the amount of IgE production and/or IL-4 production stimulated bythe native Lol p V protein allergen. Similarly, IL-4 production can becompared, with reduced IL-4 production indicating lessened IgEstimulating activity.

If a peptide of the invention is to be used as a diagnostic reagent, itis not necessary that the peptide or protein have reduced IgE bindingactivity compared to the native Lol p V allergen. IgE binding activityof peptides can be determined by, for example, using various types ofenzyme linked immunosorbent assays (ELISA).

Preferred T cell epitope containing peptide of the invention, whenadministered to a ryegrass pollen-sensitive individual or an individualsensitive to an allergen which is immunologically related to ryegrasspollen allergen such as Dac g I, in a therapeutic treatment regimen, iscapable of modifying the allergic response of the individual to theallergen. Particularly, such preferred Lol p V peptides of the inventioncomprising at least one T cell epitope of Lol p V or at least tworegions derived from Lol p V, each comprising at least one T cellepitope, when administered to an individual sensitive to ryegrass pollenare capable of modifying T cell response of the individual to theallergen and are useful as therapeutics in addressing sensitivity tograsses.

A preferred isolated Lol p V peptide of the invention comprises at leastone T cell epitope of the Lol p V and accordingly the peptide comprisesat least approximately seven amino acid residues. For purposes oftherapeutic effectiveness, preferred therapeutic compositions of theinvention preferably comprise at least two T cell epitopes of Lol p V,and accordingly, a preferred peptide comprises at least approximatelyeight amino acid residues and preferably at least fifteen amino acidresidues. Additionally, therapeutic compositions comprising preferredisolated peptides of the invention preferably comprise a sufficientpercentage of the T cell epitopes of the entire protein allergen (i.e.at least about 40% and more preferably about 60% of the T cellreactivity to the entire protein allergen) such that a therapeuticregimen of administration of the composition to an individual sensitiveto ryegrass pollen, results in T cells of the individual being tolerizedto the protein allergen. Synthetically produced peptides of theinvention comprising up to approximately forty-five amino acid residuesin length, and most preferably up to approximately thirty amino acidresidues in length are particularly desirable as increases in length mayresult in difficulty in peptide synthesis. Peptides of the invention mayalso be produced recombinantly as described earlier, and it ispreferable that peptides of 45 amino acids or longer be producedrecombinantly.

Peptides derived from the Lol p V protein allergen which can be used fortherapeutic purposes comprise at least one T cell epitope of Lol p V andcomprise all or a portion of the following peptides: LPIX-1 (SEQ IDNO:3), LPIX-1.1 (SEQ ID NO:59), LPIX-2 (SEQ ID NO:4), LPIX-2.1 (SEQ IDNO:60), LPIX-3 (SEQ ID NO:5), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ IDNO:7), LPIX-6 (SEQ ID NO:8), LPIX-7 (SEQ ID NO:9), LPIX-8 (SEQ IDNO:10), LPIX-9 (SEQ ID NO:11), LPIX-10 (SEQ ID NO:12), LPIX-11 (SEQ IDNO:13), LPIX-12 (SEQ ID NO:14), LPIX-13 (SEQ ID NO:15), LPIX-14 (SEQ IDNO:16), LPIX-15 (SEQ ID NO:17), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ IDNO:19), LPIX-18 (SEQ ID NO:20), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ IDNO:22), LPIX-21 (SEQ ID NO:23), LPIX-22 (SEQ ID NO:24), LPIX-23 (SEQ IDNO:25), LPIX-24 (SEQ ID NO:26), LPIX-26 (SEQ ID NO:28), and LPIX-27 (SEQID NO:29) (the sequences of which are shown in FIG. 2) wherein theportion of the peptide preferably has a mean T cell stimulation index(S.I.) equivalent to, or greater than the mean T cell stimulation indexof the peptide from which it is derived (e.g. as shown in FIG. 5, theS.I. for LPIX-16 (SEQ ID NO:18) is shown above the bar to be 3.7,therefore any portion of LPIX-16 preferably has a mean S.I. of 3.7).Even more preferably peptides derived from the Lol p V protein allergenwhich can be used for therapeutic purposes comprise all or a portion ofthe following peptides: LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-8 (SEQ ID NO:10), LPIX-9 (SEQ ID NO:11),LPIX-11 (SEQ ID NO:13), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18),LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22),LPIX-23 (SEQ ID NO:25), and LPIX-26 (SEQ ID NO:28) as shown in FIG. 2.Even more preferably, peptides derived from Lol p V protein allergenwhich can be used for therapeutic purposes comprise all or a portion ofthe following peptides: LPIX-1 (SEQ ID NO:3), LPIX-2 (SEQ ID NO:4),LPIX-3 (SEQ ID NO:5), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6(SEQ ID NO:8), LPIX-7 (SEQ ID NO:9), LPIX-8 (SEQ ID NO:10), LPIX-9 (SEQID NO:11), LPIX-10 (SEQ ID NO:12), LPIX-11 (SEQ ID NO:13), LPIX-12 (SEQID NO:14), LPIX-13 (SEQ ID NO:15), LPIX-14 (SEQ ID NO:16), LPIX-15 (SEQID NO:17), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-18 (SEQID NO:20), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22), LPIX-21 (SEQID NO:23), LPIX-22 (SEQ ID NO:24), LPIX-23 (SEQ ID NO:25), LPIX-24 (SEQID NO:26), LPIX-26 (SEQ ID NO:28), and LPIX-27 (SEQ ID NO:29).

One embodiment of the present invention features a peptide or portionthereof of Lol p V which comprises at least one T cell epitope of theprotein allergen and has a formula X_(n)-Y-Z_(m). According to theformula, Y is an amino acid sequence selected from the group consistingof LPIX-1 (SEQ ID NO: 3), LPIX-1.1 (SEQ ID NO:59), LPIX-2 (SEQ ID NO:4), LPIX-2.1 (SEQ ID NO:60), LPIX-3 (SEQ ID NO: 5), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO: 7), LPIX-6 (SEQ ID NO: 8), LPIX-7 (SEQ ID NO: 9),LPIX-8 (SEQ ID NO: 10), LPIX-9 (SEQ ID NO: 11), LPIX-10 (SEQ ID NO: 12),LPIX-11 (SEQ ID NO: 13), LPIX-12 (SEQ ID NO: 14), LPIX-13 (SEQ ID NO:15), LPIX-14 (SEQ ID NO: 16), LPIX-15 (SEQ ID NO: 17), LPIX-16 (SEQ IDNO: 18), LPIX-17 (SEQ ID NO: 19), LPIX-18 (SEQ ID NO: 20), LPIX-19 (SEQID NO: 21), LPIX-20 (SEQ ID NO: 22), LPIX-21 (SEQ ID NO: 23), LPIX-22(SEQ ID NO: 24), LPIX-23 (SEQ ID NO: 25), LPIX-24 (SEQ ID NO: 26),LPIX-26 (SEQ ID NO: 28), and LPIX-27 (SEQ ID NO: 29) (the sequences ofwhich are shown in FIG. 2). In addition, X_(n) are amino acid residuescontiguous to the amino terminus of Y in the amino acid sequence of theprotein allergen and Z_(m) are amino acid residues contiguous to thecarboxy terminus of Y in the amino acid sequence of the proteinallergen. In the formula, n is 0–30 and m is 0–30. Preferably, thepeptide or portion thereof has a mean T cell stimulation indexequivalent to greater than the mean T cell stimulation index of Y asshown in FIG. 4. Preferably, amino acids comprising the amino terminusof X and the carboxy terminus of Z are selected from charged aminoacids, i.e., arginine (R), lysine (K), histidine (H), glutamic acid (E)or aspartic acid (D); amino acids with reactive side chains, e.g.,cysteine (C), asparagine (N) or glutamine (Q); or amino acids withsterically small side chains, e.g., alanine (A) or glycine (G).Preferably n and m are 0–5; most preferably n+m is less than 10.

Another embodiment of the present invention provides peptides comprisingat least two regions, each region comprising at least one T cell epitopeof Lol p V and accordingly each region comprises at least approximatelyseven amino acid residues. These peptides comprising at least tworegions can comprise up to 100 or more amino acid residues butpreferably comprise at least about 14, even more preferably at leastabout 20, and most preferably at least about 30 amino acid residues ofthe Lol p V allergen. If desired, the amino acid sequences of theregions can be produced and joined by a linker to increase sensitivityto processing by antigen-presenting cells. Such linker can be anynon-epitope amino acid sequence or other appropriate linking or joiningagent. To obtain preferred peptides comprising at least two regions,each comprising at least one T cell epitope, the regions are arranged inthe same or a different configuration from a naturally-occurringconfiguration of the regions in the allergen. For example, the regionscontaining T cell epitope(s) can be arranged in a noncontiguousconfiguration and can preferably be derived from the same proteinallergen. Noncontiguous is defined as an arrangement of regionscontaining T cell epitope(s) which is different than that of the nativeamino acid sequence of the protein allergen from which the regions arederived. Furthermore, the noncontiguous regions containing T cellepitopes can be arranged in a nonsequential order (e.g., in an orderdifferent from the order of the amino acids of the native proteinallergen from which the region containing T cell epitope(s) are derivedin which amino acids are arranged from an amino terminus to a carboxyterminus). A peptide of the invention can comprise at least 15%, atleast 30%, at least 50% or up to 100% of the T cell epitopes of Lol p Vbut does not comprise the entire amino acid sequence of Lol p V.

The individual peptide regions can be produced and tested to determinewhich regions bind immunoglobulin E specific for Lol p V and which ofsuch regions would cause the release of mediators (e.g., histamine) frommast cells or basophils. Those peptide regions found to bindimmunoglobulin E and to cause the release of mediators from mast cellsor basophils in greater than approximately 10–15% of the allergic seratested are preferably not included in the peptide regions arranged toform preferred peptides of the invention.

Examples of preferred peptide regions which do not appear to bind to IgEin preliminary IgE binding data studies (Example 3) include the aminoacid sequences of such regions being shown in FIG. 2 (SEQ ID NO:3–29),or portions of said regions comprising at least one T cell epitope.

Preferred peptides comprise various combinations of two or more of theabove-discussed preferred regions, or a portion thereof. Preferredpeptides comprising a combination of two or more regions (each regionhaving an amino acid sequence as shown in FIG. 2), include thefollowing:

LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ IDNO:22);

LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22);

LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO: 8),LPIX-17 (SEQ ID NO:19) and LPIX-20 (SEQ ID NO:22);

LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8) andLPIX-20 (SEQ ID NO:22);

LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-11 (SEQ ID NO:13), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18),LPIX-17 (SEQ ID NO:19) and LPIX-20 (SEQ ID NO:22);

LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-8(SEQ ID NO:10), LPIX-9 (SEQ ID NO:11), LPIX-11 (SEQ ID NO:13), LPIX-12(SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19(SEQ ID NO:21), LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ ID NO:25) andLPIX-26 (SEQ ID NO:28);LPIX-4 (SEQ ID NO:6), LPIX-1 I (SEQ ID NO: 13), LPIX-16 (SEQ ID NO:18),and LPIX-20 (SEQ ID NO:22);LPIX-4 (SEQ ID NO:6), LPIX-11 (SEQ ID NO: 13), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22);LPIX-4 (SEQ ID NO:6), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22);LPIX-5 (SEQ ID NO:7), LPIX-11 (SEQ ID NO: 13), LPIX-16 (SEQ ID NO:18),and LPIX-20 (SEQ ID NO:22);LPIX-5 (SEQ ID NO:7), LPIX-11 (SEQ ID NO: 13), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22);LPIX-5 (SEQ ID NO:7), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22);LPIX-11 (SEQ ID NO: 13), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22);LPIX-4 (SEQ ID NO:6), LPIX-11 (SEQ ID NO: 13), and LPIX-20 (SEQ IDNO:22);LPIX-4 (SEQ ID NO:6), LPIX-16 (SEQ ID NO:18), and LPIX-20 (SEQ IDNO:22);LPIX-4 (SEQ ID NO:6), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ IDNO:22);LPIX-5 (SEQ ID NO:7), LPIX-11 (SEQ ID NO: 13), and LPIX-20 (SEQ IDNO:22);LPIX-5 (SEQ ID NO:7), LPIX-16 (SEQ ID NO:18), and LPIX-20 (SEQ IDNO:22);LPIX-11 (SEQ ID NO: 13), LPIX-16 (SEQ ID NO:18), and LPIX-20 (SEQ IDNO:22);LPIX-11 (SEQ ID NO: 13), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ IDNO:22);LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ IDNO:22).

In yet another aspect of the present invention, a composition isprovided comprising at least two peptides (e.g., a physical mixture ofat least two peptides), each comprising at least one T cell epitope ofLol p V. Such compositions can be in the form of a compositionadditionally with a pharmaceutically acceptable carrier of diluent fortherapeutic uses, or with conventional non-pharmaceutical excipients forreagent use. When used therapeutically, an effective amount of one ormore of such compositions can be administered simultaneously orsequentially to an individual sensitive to ryegrass pollen.

In another aspect of the invention, combinations of Lol p V peptides areprovided which can be administered simultaneously or sequentially. Suchcombinations may comprise therapeutic compositions comprising only onepeptide, or more peptides if desired. Such compositions may be usedsimultaneously or sequentially in preferred combinations.

Preferred compositions and preferred combinations of Lol p V peptideswhich can be administered or otherwise used simultaneously orsequentially (comprising peptides having amino acid sequences shown inFIG. 2) include the following combinations:

LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ IDNO:22);

LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22);

LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-17 (SEQ ID NO:19) and LPIX-20 (SEQ ID NO:22);

LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8) andLPIX-20 (SEQ ID NO:22);

LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-11 (SEQ ID NO: 13), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18),LPIX-17 (SEQ ID NO:19) and LPIX-20 (SEQ ID NO:22);

LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-8(SEQ ID NO:10), LPIX-9 (SEQ ID NO:11), LPIX-11 (SEQ ID NO:13), LPIX-12(SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19(SEQ ID NO:21), LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ ID NO:25) andLPIX-26 (SEQ ID NO:28);LPIX-4 (SEQ ID NO:6), LPIX-11 (SEQ ID NO: 13), LPIX-16 (SEQ ID NO:18),and LPIX-20 (SEQ ID NO:22);LPIX-4 (SEQ ID NO:6), LPIX-11 (SEQ ID NO: 13), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22);LPIX-4 (SEQ ID NO:6), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22);LPIX-5 (SEQ ID NO:7), LPIX-11 (SEQ ID NO: 13), LPIX-16 (SEQ ID NO:18),and LPIX-20 (SEQ ID NO:22);LPIX-5 (SEQ ID NO:7), LPIX-11 (SEQ ID NO: 13), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22);LPIX-5 (SEQ ID NO:7), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22);LPIX-11 (SEQ ID NO: 13), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22);LPIX-4 (SEQ ID NO:6), LPIX-11 (SEQ ID NO: 13), and LPIX-20 (SEQ IDNO:22);LPIX-4 (SEQ ID NO:6), LPIX-16 (SEQ ID NO:18), and LPIX-20 (SEQ IDNO:22);LPIX-4 (SEQ ID NO:6), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ IDNO:22);LPIX-5 (SEQ ID NO:7), LPIX-11 (SEQ ID NO: 13), and LPIX-20 (SEQ IDNO:22);LPIX-5 (SEQ ID NO:7), LPIX-16 (SEQ ID NO:18), and LPIX-20 (SEQ IDNO:22);LPIX-11 (SEQ ID NO: 13), LPIX-16 (SEQ ID NO:18), and LPIX-20 (SEQ IDNO:22);LPIX-11 (SEQ ID NO: 13), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ IDNO:22);LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ IDNO:22).

In another aspect of the present invention, a therapeutic composition isprovided comprising at least two peptides (e.g. a physical mixture of atleast two peptides, each peptide comprising at least one epitope)wherein at least one peptide, comprises an amino acid sequence orportion thereof derived from Lol p V selected from the following group:LPIX-1 (SEQ ID NO:3), LPIX-2 (SEQ ID NO:4), LPIX-3 (SEQ ID NO:5), LPIX-4(SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-7 (SEQID NO:9), LPIX-8 (SEQ ID NO:10), LPIX-9 (SEQ ID NO:11), LPIX-10 (SEQ IDNO:12), LPIX-11 (SEQ ID NO:13), LPIX-12 (SEQ ID NO:14), LPIX-13 (SEQ IDNO:15), LPIX-14 (SEQ ID NO:16), LPIX-15 (SEQ ID NO:17), LPIX-16 (SEQ IDNO:18), LPIX-17 (SEQ ID NO:19), LPIX-18 (SEQ ID NO:20), LPIX-19 (SEQ IDNO:21), LPIX-20 (SEQ ID NO:22), LPIX-21 (SEQ ID NO:23), LPIX-22 (SEQ IDNO:24), LPIX-23 (SEQ ID NO:25), LPIX-24 (SEQ ID NO:26), LPIX-26 (SEQ IDNO:28), and LPIX-27 (SEQ ID NO:29) (as shown in FIG. 2), and wherein atleast one peptide comprises an amino acid sequence or portion thereofderived from Lol p I selected from the following group: LPI-1 (SEQ IDNO:30), LPI-1.1 (SEQ ID NO:31), LPI-2 (SEQ ID NO:32), LPI-3 (SEQ IDNO:55), LPI-4 (SEQ ID NO:33), LPI-4.1 (SEQ ID NO:34), LPI-5 (SEQ IDNO:35), LPI-6 (SEQ ID NO:36), LPI-7 (SEQ ID NO:37), LPI-8 (SEQ IDNO:38), LPI-9 (SEQ ID NO:39), LPI-10 (SEQ ID NO:40), LPI-11 (SEQ IDNO:41), LPI-12 (SEQ ID NO:42), LPI-13 (SEQ ID NO:43), LPI-14 (SEQ IDNO:44), LPI-15 (SEQ ID NO:45), LPI-16 (SEQ ID NO:46), LPI-16.1 (SEQ IDNO:47), LPI-17 (SEQ ID NO:48), LPI-18 (SEQ ID NO:49), LPI-19 (SEQ IDNO:50), LPI-20 (SEQ ID NO:56), LPI-21 (SEQ ID NO:51), LPI-22 (SEQ IDNO:52), and LPI-23 (SEQ ID NO:53) (as shown in FIG. 3). The isolationand cloning of the clones encoding Lol p I as well as the synthesis ofthe various Lol p I peptides shown in FIG. 3, along with human T cellstudies using Lol p I and using various peptides derived from Lol p Iare described in PCT/US94/02537, which is hereby incorporated byreference in its entirety.

Preferably, a therapeutic composition comprises at least five, six,seven, or eight peptides wherein at least three or four peptides arederived from Lol p V and are selected from the following group: LPIX-4(SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-11 (SEQ ID NO: 13), LPIX-16(SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ ID NO:22), andat least two, three or four peptides are derived from Lol p I andselected from the following group: LPI-16 (SEQ ID NO:46), LPI-18 (SEQ IDNO:49), LPI-20 (SEQ ID NO:56), and LPI-23 (SEQ ID NO:53); for example, apreferred therapeutic composition comprises at least two peptides of Lolp I and three peptides of Lol p V, or three peptides from Lol p I andthree peptides from Lol p V, or three peptides from Lol p I and fourpeptides from Lol p V, or four peptides from Lol p I and four peptidesfrom Lol p V, or four peptides from Lol p I and three peptides from Lolp V.

In another aspect of the present invention a method is providedcomprising administering a combination of peptides or portions thereofderived from Lol p V and Lol p I which can be administeredsimultaneously or sequentially; each of such peptides can be in the formof a therapeutic composition with a pharmaceutically acceptable carrieror diluent. Examples of preferred compositions and preferredcombinations comprising Lol p V and Lol p I peptides or portionsthereof, which can be administered simultaneously or sequentiallycomprise the following combinations:

LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45),LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-8 (SEQ ID NO:10), LPIX-9 (SEQ ID NO:11),LPIX-11 (SEQ ID NO:13), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18),LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22),LPIX-23 (SEQ ID NO:25), LPIX-26 (SEQ ID NO:28);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20, LPI-23 (SEQ IDNO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34), LPI-10 (SEQ IDNO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45), LPI-22 (SEQ IDNO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ IDNO:8), LPIX-8 (SEQ ID NO:10), LPIX-9 (SEQ ID NO:11), LPIX-11 (SEQ IDNO:13), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ IDNO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ IDNO:25), LPIX-26 (SEQ ID NO:28), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ IDNO:7), LPIX-6 (SEQ ID NO:8), LPIX-9 (SEQ ID NO:11), LPIX-11 (SEQ IDNO:13), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ IDNO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45),LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-9 (SEQ ID NO:11), LPIX-12 (SEQ ID NO:14),LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21),LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ ID NO:25);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45),LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18),LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45),LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45),LPI-22 (SEQ ID NO:52);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45),LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-8(SEQ ID NO:10), LPIX-9 (SEQ ID NO:11), LPIX-11 (SEQ ID NO:13), LPIX-12(SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19(SEQ ID NO:21), LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ ID NO:25), LPIX-26(SEQ ID NO:28);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID: NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45),LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-9(SEQ ID NO:11), LPIX-11 (SEQ ID NO:13), LPIX-12 (SEQ ID NO:14), LPIX-16(SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20(SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45),LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-9(SEQ ID NO:11), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17(SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45),LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-12 (SEQ ID NO:14), LPIX-16 SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45),LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21),LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-10 (SEQ ID NO:40), LPI-11 (SEQ ID NO:41), LPI-15 (SEQ ID NO:45),LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-8(SEQ ID NO:10), LPIX-9 (SEQ ID NO:11), LPIX-11 (SEQ ID NO:13), LPIX-12(SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19(SEQ ID NO:21), LPIX-20 (SEQ ID NO:22); LPIX-23 (SEQ ID NO:25), LPIX-26(SEQ ID NO:28);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-9(SEQ ID NO:11), LPIX-11 (SEQ ID NO:13), LPIX-12 (SEQ ID NO:14), LPIX-16(SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20(SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-9(SEQ ID NO:11), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17(SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22), LPIX-23(SEQ ID NO:25);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21),LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-22 (SEQ. ID NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-8 (SEQ. ID NO:10), LPIX-9 (SEQ. ID NO:11),LPIX-11 (SEQ. ID NO:13), LPIX-12 (SEQ. ID NO:14), LPIX-16 (SEQ IDNO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ. ID NO:21), LPIX-20 (SEQ IDNO:22), LPIX-23 (SEQ. ID NO:25), LPIX-26 (SEQ. ID NO:28);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-8 (SEQ ID NO:10), LPIX-9 (SEQ ID NO:11),LPIX-11 (SEQ ID NO:13), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18),LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-9 (SEQ ID NO:11), LPIX-12 (SEQ ID NO:14),LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21),LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ ID NO:25);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18),LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPI-4.1 (SEQ ID NO:34),LPI-22 (SEQ ID NO:52), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPI-3 (SEQ ID NO:55), LPIX-4 (SEQ ID NO:6),LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-8 (SEQ ID NO:10),LPIX-9 (SEQ ID NO:11), LPIX-11 (SEQ ID NO:13), LPIX-12 (SEQ ID NO:14),LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21),LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ ID NO:25), LPIX-26 (SEQ ID NO:28);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-9 (SEQ ID NO:11), LPIX-11 (SEQ ID NO:13),LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-9 (SEQ ID NO:11), LPIX-12 (SEQ ID NO:14),LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21),LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ ID NO:25);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18),LPIX-17 (SEQ ID NO:19), LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22);LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56),LPI-23 (SEQ ID NO:53), LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),LPIX-19 (SEQ ID NO:21), LPIX-20 (SEQ ID NO:22); and--.LPI-16.1, LPI-18, LPI-20, LPI-23, LPIX-4, LPIX-5, LPIX-6, LPIX-16,LPIX-17, LPIX-20 with --LPI-16.1 (SEQ ID NO:47), LPI-18 (SEQ ID NO:49),LPI-20 (SEQ ID NO:56), LPI-23 (SEQ ID NO:53), LPIX-4 (SEQ ID NO:6),LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-16 (SEQ ID NO:18),LPIX-17 (SEQ ID NO:19), LPIX-20 (SEQ ID NO:22).

In addition, a composition is provided comprising at least two Lol p Ipeptides (e.g. a physical mixture of at least two peptides), eachcomprising at least one T cell epitope of Lol p I. Such compositions canbe administered in the form of a therapeutic composition with with apharmaceutically acceptable carrier or diluent to treat ryegrasssensitivity and particularly, sensitivity to Lol p I protein allergen.Preferred compositions and preferred combinations of Lol p I peptideswhich can be administered simultaneously or sequentially (comprisingpeptides having the amino acid sequences shown in FIG. 3 include thefollowing combinations:

LPI-16 (SEQ ID NO:46), and LPI-20 (SEQ ID NO:56);

LPI-18 (SEQ ID NO:49), and LPI-20 (SEQ ID NO:56);

LPI-20 (SEQ ID NO:56), and LPI-23 (SEQ ID NO:53);

LPI-16 (SEQ ID NO:46), LPI-18 (SEQ ID NO:49), and LPI-20 (SEQ ID NO:56);

LPI-16 (SEQ ID NO:46), LPI-20 (SEQ ID NO:56), and LPI-23 (SEQ ID NO:53);

LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), and LPI-23 (SEQ ID NO:53);

LPI-16 (SEQ ID NO:46), LPI-18 (SEQ ID NO:49), LPI-20 (SEQ ID NO:56), andLPI-23 (SEQ ID NO:53).

Any of the compositions described herein are useful in the manufactureof a medicament for treating sensitivity to ryegrass pollen allergen oran immunologically cross reactive allergen in an individual.

The present invention if further illustrated by the followingnon-limiting Figures and Examples.

EXAMPLE I

Purification of Native Lol p V from Ryegrass Pollen

A. Production and Purification of Monoclonal Antibody (mAb) 1B9.

Balb/c mice were immunized with crude Dactylis glomerata (orchardgrass/cocksfoot grass) pollen extract and antibody secreting clones weregenerated as described (Walsh et al., Int. Arch. Allergy Appl. Immunol.,1990, 91: 419–425). MAb 1B9 hybridoma clone which cross-reacts to Lol pV was obtained from Dr. Walker (Univ. Birmingham, Wolfson Research Lab,Birmingham, UK). Ascitis fluid generated from Balb/c mice was producedby contract (Babco, Richmond, Calif.). The antibodies were purified fromascites fluid by (NH₄)₂ SO₄ precipitation (50% saturation). The pelletwas resuspended in 10 mM phosphate buffer, pH 7.5 and dialyzed againstthe same buffer at 4° C. overnight and then fractionated by ion-exchangechromatography on FPLC Q-SEPHAROSE column (Pharmacia, Piscataway, N.J.)using linear gradient 0–0.5 M NaCl. IgG was eluted between 0.15–0.2 MNaCl concentration.

B. Preparation of 1B9 Immunoaffinity Column

Purified 1B9 was coupled to AFFIGEL-10 resin (Biorad, Richmond, Calif.)using 3–4 mg protein/mL of gel according to manufacturer's instructions.In brief, PFLC Q-SEPHAROSE column purified mAb 1B9 was dialyzed against0.1M MOPS buffer, pH 7.5 with two to three changes overnight at 4° C.The AFFIGEL-10 resin was washed with deionized cold H₂O in a scinteredglass funnel. The washed resin was mixed with the 1 B9 antibody for fourhours at 4° C., followed by an one-hour blocking step with 1 Methanolamine, pH 8.0. Resin was packed into a column, washed with PBSand than stored in PBS+0.05% sodium azide.

C. Affinity Purification of Lol p V from Ryegrass Pollen

100 g defatted ryegrass pollen (purchased from Greer Laboratories,Lenoir, N.C.) was extracted 1 liter extraction buffer containing 0.05 Mphosphate buffer, pH 7.2, 0.15 M NaCl, phenyl methyl sulfonyl fluoride(170 μg/mL), leupeptin (1 μg/mL), pepstatin (1 μg/mL) and soybeantrypsin inhibito (1 μg/mL).

The pollen was extracted by stirring the solution overnight at 4° C.,followed by centrifugation 12,000×g for 100 minutes. The insolublematerials were re-extracted in 0.5–1.0L extraction buffer a then thesupernatants were combined and depigmented by batch absorption onto 100mL DE-52 cellulose (Whatman, Maidstone, England) equilibrated with 0.05M phosphate buffer+0.3 M NaCl, 7.2.

The unbound materials were loaded onto the 1B9 AFFIGEL-10 column at aflow rate of 0.5 ml/min. The column was then washed extensively withPBS, PBS+0.5 NaCl and once again with PBS before elution of the Lol p Vallergens with 0.1 M glycine, pH 2.7. Fractions were neutralized with 1M Tris, pH 11.0 immediately. These affinity-purified materials were usedin IgE studies and T cell epitope mapping.

Physicochemical Properties of Affinity-Purified Lol p V

The 1B9 affinity-purified material was analyzed by SDS-PAGE. As shown inFIG. 15, Lol p exists as multiple bands with molecular weight rangedfrom 29,000–22,000. All these components were reactive with 1B9 byWestern blotting analysis (data not shown). These components wereelectroblotted onto ProBlott membrane (Applied Biosystems, Foster City,Calif.), stained by Coomassi blue and the three major bands were excisedand sequenced on a Beckman LF-3000 sequencer (Beckman Instruments,Carlsbad, Calif.). N-terminal amino acid sequence of the three bands areshow Table I. The sequencing data shows that the middle and lowermolecular weight bands represent N-terminal cleavage products of thehigher molecular weight component. The N-terminus sequence wa identicalto the cloned Lol p V (12R) (see PCT application publication numberWO93/04174). The 5 proline residues at the N-terminus were found to beall hydroxyprolines, which seemed to be commo Group V allergens fromNorthern grasses (Matthiesen, F. et al., 1991, Clin. Exp. Allergy,21:297–307 We also determined the 1B9-affinity purified material byamino acid analysis (Table 2) and the data were very similar to the Lolp V and other group V allergens from Northern grasses reported by Klyset al., (Clin. Experimental Allergy, 1992, 22:491–497). Furthermore,Western blot analysis using specific anti-group I mAb (data not shown)demonstrated Group I proteins could not be detected in th preparations.Thus, taken together these data suggest that the 1B9-affinity purifiedpreparations contained only Group V allergens.

TABLE 1 N-terminal amino acid sequence and cleavage site of Lol p Vallergen amino acid # 1         11 Lol p V (SEQ ID NO:54)ADAGYTP′AAAATP′ATP′AATP′ 21        31 AAAGGKATTDEQK P′ representshydroxyproline The N-terminal sequence was determined from the threemajor bands electroblotted onto ProBlott membrane. The upper band startswith amino acid 1 whereas the middle and the lower bands start at aminoacid 9 and 18, respectively. The arrows indicate the cleavage sites.

TABLE 2 Amino acid composition of Group V allergens Mole % Lol p V^(b)Amino acid Phl p V^(a) Lol p V^(a) expt 1 expt 2 expt 3 Asx 5.4 6.3 5.36.7 7.5 Thr 7.6 8.6 7.4 8.7 9.2 Ser 5.1 2.0 3.3 2.3 2.7 Glx 10.2 9.8 7.48.8 8.9 Gly 6.4 4.0 7.2 5.2 4.8 Ala 25.7 29.0 27.7 31.3 31.7 Cys 0.0 1.0— — — Val 6.6 6.4 5.5 5.5 6.4 Met 0.7 0.3 0.5 0.3 0.8 Ile 3.6 3.4 3.52.9 3.1 Leu 4.7 5.9 6.5 5.0 5.3 Tyr 3.5 3.0 2.9 2.5 1.7 Phe 4.1 5.0 4.84.0 4.5 His 0.8 0.3 — 0.2 0.5 Lys 8.8 9.8 11.0 9.2 6.0 Arg 1.0 0.4 0.60.4 0.8 Pro 4.5 4.9 5.4^(c) 4.7^(c) 3.7^(c) Hyp 1.4 N.R. 1.5^(c) 1.8^(c)1.7^(c) N.R. (Not reported) ^(a)values reported by Klysner, S. et al.Clin. Exp. Allergy (1992) 22: 491–497. ^(b)the amino acid compositionwas determined from mAb 1B9-affinity purified materials and valuesobtained from three experiments are presented. ^(c)the content ofproline and hydroxyproline was determined by peak height since thehydroxyproline peak was very broad due to an contaminant which eluted atthe trailing edge of the hydroxyproline peak. All the other amino acidswere determined by peak areas.

EXAMPLE 2 Human T Cell Studies with Lol p V

Synthesis of Overlapping Peptides

The amino acid sequence of Lol p V was deduced from the cDNA sequence ofclone 12R (SEQ ID NO:2) ATCC number 69475 as shown in FIG. 1. Thedetails of the isolation and cloning of clone 12R encoding Lol p V(described as Lol p Ib.1) are given in PCT application publicationnumber WO93/04174 incorporated herein by reference in its entirety. Oneexample of expression of recombinantly produced Lol p V encoded by clone12R is given in Example 4, to follow.

Ryegrass Lol p V overlapping peptides were synthesized using standardFmoc/tBoc synthetic chemistry and purified by Reverse Phase HPLC. FIG. 2shows Lol p V peptides used in these studies. The peptide names areconsistent throughout.

T Cell Responses to Ryegrass Antigen Peptides

Peripheral blood mononuclear cells (PBMC) were purified by lymphocyteseparation medium (LSM) centrifugation of 60 ml of heparinized bloodfrom grass-allergic patients who exhibited clinical symptoms of seasonalrhinitis and were skin test positive for grass. Long-term T cell lineswere established by stimulation of 2×10 ⁶ PBL/ml in bulk cultures ofcomplete medium (IRPMI-164), 2 mM L-glutamine, 100 U/mlpenicillin/streptomycin, 5×10⁻⁵M 2-mercaptoethanol, and 10 mM HEPES,supplemented with 5% heat-inactivated human AB serum, with 10 μg/ml ofaffinity purified native Lol p V for 6 days at 37° C. in a humidified 5%CO₂ incubator to select for Lol p V reactive T Cells. This amount ofpriming antigen was determined to be optimal for the activation of Tcells from most grass-allergic patients. Viable cells were purified byLSM centrifugation and cultured in complete medium, supplemented with 5units recombinant human IL-2/ml and 5 units recombinant human IL-4/mlfor up to 3 weeks until the desired cell number were achieved. The cellswere allowed to rest for 4–6 days.

The ability of the T cells to proliferate to selected peptides,recombinant Lol p I (rLol p I), purified native Lol p V, purified rLol pV, or recombinant Fel d I (rFel d I) (chain I), or tetanus toxoid (TT)was then assessed. For assay, 2×10⁴ rested cells were restimulated inthe presence of 2×10⁴ autologous Epstein-Barr virus (EBV)-transformed Bcells (prepared as described below) or 5×10⁴ irradiated PBL with 2–50mg/ml of rLol p I, purified native Lol p V, rFel d I (Chain I), or rLolp I, in a volume of 200 ml complete medium in duplicate wells in 96-wellround-bottom plates for three days. Each well then received 1 mCitritiated thymidine for 16–20 hours. The counts incorporated werecollected onto glass fiber filter mats and processed for liquidscinitillation counting. The varying antigen dose in assays with rLol pV, purified native Lol p V, and recombinant Lol p I and antigenicpeptides synthesized as described above were determined. The titrationswere used to optimize the dose of peptides in T cell assays. The maximumresponse in a titration of each peptide is expressed as the stimulationindex (S.I.). The S.I. is the counts per minute (CPM) incorporated bycells in response to peptide, divided by the CPM incorporated by cellsin medium only. An S.I. value equal to or greater than 2 times thebackground level is considered “positive” and indicates that the peptidecontains a T cell epitope. The positive results were used in calculatingmean stimulation indices for each peptide for the group of patientstested. The results (not shown) demonstrate that one patient respondswell to recombinant Lol p V and purified native Lol p V, as well as toLol p V peptides but not to rFel d I (Chain I) or TT. This indicatedthat Lol p V T cell epitopes are recognized by T cells from thisparticular allergic patient and that rLol p V contains such T cellepitopes.

The above procedure was followed with a total of 19 patients. Individualpatient results were used in calculating the mean S.I. for each peptideif the patient responded to the purified native Lol p V protein at anS.I. of 2.0 or greater and the patient responded to at least one peptidederived from purified native Lol p I at an S.I. of 2.0 or greater. Asummary of positive experiments from 19 patients is shown in FIG. 4. Thenumbers above each bar report the mean S.I. for that peptide. Thenumbers enclosed in the parentheses denote percentage of patientsresponding to that particular peptide. The bar represents the positivityindex for each peptide (% of patients responding multiplied by meanS.I.).

FIG. 5 shows the ranked sum for each peptide derived from the same dataas described above. The bar represents the cumulative rank of thepeptide response in the group of the 19 patients tested. To determinethe cumulative rank, the 5 peptides with the highest S.I. in eachindividual are determined and assigned a numerical rank in descendingorder, with 5 representing the strongest response. The ranks for eachpeptide were then summed for the entire group of patients to determinethe cumulative rank for the peptide. Above each bar is the mean S.I. andpercent of positive responses (in parenthesis) with an S.I. of at least2 to the peptide in the group of 19 patients tested. Given the percentpositive and the mean T cell stimulation index, the positivity index(P.I.) for each peptide can be calculated by multiplying the twonumbers. FIG. 5 shows that LPIX-20 has the highest ranked sum of thepeptides in this study.

EXAMPLE 3

Lol p V as a Major Ryegrass Pollen Allergen

A) ELISA Analysis

To examine the importance of Lol p V, both direct and competition ELISAassays were performed. In the direct ELISA, 100 μl of 10 μg/ml ofantigen in Phosphate Buffered Saline, pH 7.4 (PBS) was used to coatImmulon II (Dynatech, Chantilly, Va.) 96 well plates for 4 hours at roomtemperature (RT) or overnight (O/N) at 4° C. In between each step theplates were washed 3× with PBS-T. The excess coating antigen(s) wasremoved and the wells blocked with 300 μl/well 0.5% gelatin +1 mg/ml PVPin PBS for 1 hour at RT. Serially diluted patient plasma or the diluentPBS+0.05% Tween⁻²⁰ was incubated in at 100 μl/well in duplicate wellsovernight at 4° C. Unbound antibody was removed, and the wells incubatedwith 100 l/well of 2nd Ab (1:1000, biotinylated goat anti-human IgE, KPLInc., Gaithersburg, Md.) for 1 hour at RT. This solution was removed andstreptavidin-horse radish peroxidase (HRPO) (1:10000) was added at 100μl/well (SBA Inc., Birmingham, Ala.) and incubated for 1 hr at RT. 3,3′, 5,5′-tetramethylbenzidine (TMB) Substrate (KPL, Gaithersburg, Md.)was freshly mixed and added at 100 μl/well and the color allowed todevelop for 1–5 minutes. The reaction was stopped by the addition of 100μl/well 1M phosphoric acid. Plates were read on a MR7000 plate reader(Dynatech, Chantilly, Va.) with a 450 nm filter. The absorbance levelsof duplicate wells were averaged. The results were graphed as absorbancevs. dilution. The competition ELISA were carried out using the sameprotocol with the following changes: a single dilution of patient plasma(or pooled human plasma (PHP)) was used as the source of IgE; seriallydiluted antigen was mixed with the plasma and allowed to incubate O/N at4° C. This plasma was then incubated on duplicate wells. The results areplotted as the absorbance vs. the log of the concentration of competingantigen.

For the direct ELISA, wells were coated with either soluble pollenextract (SPE) of ryegrass pollen or rLol p V (purified native Lol p Vmay have a small amount of Lol p I; use of recombinant material assuresthat the IgE binding is only to Lol p V) and human IgE antibody bindingto these antigens was analyzed. PHP, consisting of an equal volume ofplasma from 20 patients with a ryegrass prick test score of 3+ orgreater (PHP-A), or PHP consisting of equal aliquots of plasma from 40grass skin test reactive patients with high IgE binding as measured bydirect ELISA (PHP-B), or plasma from individual patients were comparedin this assay. The results of binding reactivity with PHP-A (FIG. 6),PHP-B (FIG. 7), four individual patients on ryegrass pollen SPE (FIG.8), and purified rLol p V (FIG. 9) to either SPE or rLol p V, indicatethat there is high IgE binding to both the pollen extract and therecombinant protein.

In the competition assay, ELISA wells were coated with ryegrass pollenSPE and then allergic patient IgE binding was measured in the presenceof competing ryegrass pollen SPE, purified native Lol p V, or rLol p V.The source of allergic IgE in this assay was PHP-A (FIG. 10) orindividual patient plasma (FIG. 11). The competition assays confirm thata significant portion of IgE against Lol p SPE is specific for Lol p V.

B) Histamine Release Analysis

A histamine release assay was performed on one ryegrass allergicindividual, using Lol p SPE and rLol p V as the added antigens. Thisassay is a measure of IgE reactivity through human basophil mediatorrelease, and it is based on the detection of an acylated derivative ofhistamine using a specific monoclonal antibody (Morel, A. M. andDelaage, M. A.; 1988, J. Allergy Clin. Immunol. 82: 646–654). Thereagents for this radioimmunoassay are sold as a kit by Amac Inc.(Westbrook, Me.). Whole heparinized blood drawn from a grass allergicindividual and then 20011 aliquots were mixed with an equal amount ofthe grass antigens SPE and rLol p V at various concentrations or thediluent, PACM buffer (25 mM PIPES, 100 mM NaCl, 5 mM KCL, 4 mM CaCl₂, 1mM MgCl₂, 0.003% HSA, pH7.3) in 1.5 ml polypropylene. The releasereactions were carried out at 37° C. for 30 minutes. After thisincubation the samples were centrifuged at 1500 RPM for 3 minutes andthe supernatants removed. For the total histamine release, 0.1 ml ofblood was added to 0.9 ml of PACM buffer, vortexed, and then boiled for3 minutes. The samples were spun at 13000 RPM and the supernatantremoved for analysis. Duplicate samples were used to measure totalrelease. All of the supernatants are diluted 1:4 in acylation buffer andthe remainder of the assay is performed according to the manufacturer'sinstructions. The results of this assay, shown in FIG. 12, demonstratestrong histamine release over a wide concentration range for both theextract and the recombinant protein.

C) Reactivity to Lol p V Peptides

Direct ELISA was performed to assess the IgE reactivity to Lol p Vpeptides. In this assay ELISA plates were coated with the set ofsynthetic Lol p V peptides (as shown in FIG. 2) and rLol p V protein.Human IgE binding of PHP-B was incubated on the wells and the resultingbinding analyzed. As evidenced in FIG. 13 a and FIG. 13 b there is nosignificant binding detected to any of the Lol p V peptides in thispreliminary assay although there is very high IgE binding to Lol p Vprotein.

D) Lol p I and Lol p V Constitute the Major Allergens of Ryegrass Pollen

A separate competition ELISA was done to show that Lol p I and Lol p Vtogether constitute the major IgE binding proteins of ryegrass pollenSPE. In this assay (FIG. 14), PHP-B was used to examine the ability of amixture of native purified Lol p I and Lol p V or a mixture of rLol p Iand rLol p V to compete for IgE binding to ryegrass pollen SPE. Themixture of purified native proteins competes to background level the IgEbinding to ryegrass pollen SPE. The mixture of rLol p I and rLol p V isalso able to substantially reduce the amount of IgE available to bind tothe SPE coating the plate. The majority of human IgE directed againstall of the ryegrass pollen proteins was bound up by the mix of just twoproteins (Lol p I and Lol p V) found in the complex mix of ryegrasspollen SPE proteins. This data implies that these two proteins are majorallergens of ryegrass pollen.

EXAMPLE 4

Expression of Lol p V

Expression of Lol p V was performed as follows. The λgtII clone 12R wasdigested with EcoRI. The insert encoding Lol p V was ligated into pGEX.A pGEX vector containing Lol p V (clone 12R) was digested with EcoRI.The Lol p V insert (containing the nucleotide sequence shown in FIG. 1)was isolated by electrophoresis of this digest through a 1% SeaPlaquelow melt agarose gel. The insert was then ligated into EcoRI digestedexpression vector pET-11d (Novagen, Madison, Wis.; Jameel et al. (1990)J. Virol. 64:3963–3966) modified to contain a sequence encoding 6histidines (His 6) immediately 3′ of the ATG initiation codon followedby a unique EcoR I endonuclease restriction site. A second EcoR Iendonuclease restriction site in the vector, along with neighboring ClaI and Hind III endonuclease restriction sites, had previously beenremoved by digestion with EcoR I and Hind III, blunting and religation.The histidine (His₆) sequence was added for affinity purification of therecombinant protein (rLol p V) on a Ni²⁺ chelating column (Hochuli etal. (1987) J. Chromatog. 411:177–184; Hochuli et al. (1988) Bio/Tech.6:1321–1325.). A recombinant clone was used to transform Escherichiacoli strain BL21-DE3 which harbors a plasmid that has anisopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter precedingthe gene encoding T7 polymerase. Induction with IPTG leads to highlevels of T7 polymerase expression, which is necessary for expression ofthe recombinant protein in pET-11d, which has a T7 promoter. The pET-11dcontaining the Lol p V (clone 12R) was confirmed by dideoxy sequencing(Sanger et al., (1977) Proc. Natl. Acad. Sci., (USA) 74:5460–5463) to bea Lol p V clone in the correct reading frame for expression.

The pET-11d Lol p V clone was grown on a large scale for recombinantprotein expression and purification. A 2 ml culture of bacteriacontaining the recombinant plasmid was grown for 8 hr, then streakedonto solid media (e.g. 6 petri plates (100×15 mm) with 1.5% agarose inLB medium (Gibco-BRL, Gaithersburg, Md.) containing 200 μg/mlampicillin), grown to confluence overnight, then scraped into 9 L ofliquid media (Brain Heart Infusion media, Difco) containing ampicillin(200 μg/ml). The culture was grown until the A₆₀₀ was 1.0, IPTG added (1mM final concentration), and the culture grown for an additional 2hours.

Bacteria were recovered by centrifugation (7,930×g, 10 min), and lysedin 90 ml of 6M Guanidine-HCl, 0.1 M Na₂HPO₄, pH 8.0 for 1 hour withvigorous shaking. Insoluble material was removed by centrifugation(11,000×g, 10 min, 4° C.). The pH of the lysate was adjusted to pH 8.0,and the lysate applied to an 80 ml Nickel NTA agarose column (Qiagen,Chatsworth, Calif.) that had been equilibrated with 6 M Guanidine HCl,100 mM Na₂HPO₄, pH 8.0. The column was sequentially washed with 6 MGuanidine HCl, 100 mM Na₂HPO₄, 10 mM Tris-HCl, pH 8.0, then 8 M urea,100 mM Na₂HPO₄, pH 8.0, and finally 8 M urea, 100 mM sodium acetate, 10mM Tris-HCl, pH 6.3. The column was washed with each buffer until theflow through had an A₂₈₀<0.05.

The recombinant protein, rLol p V, was eluted with 8 urea, 100 mM sodiumacetate, 10 mM Tris-HCl, pH 4.5, and collected in 10 ml aliquots. Theprotein concentration of each fraction was determined by absorbance atA₂₈₀ and the peak fractions pooled. An aliquot of the collectedrecombinant protein was analyzed on SDS-PAGE (data not shown) accordingto the method in Sambrook et al., supra.

The first 9 liter preparation yielded 12 mg of rLol p V withapproximately 60–70% purity. Purity of the preparation was determined bydensitometry (Shimadzu Flying Spot Scanner, Shimadzu ScientificInstruments, Inc., Braintree, Mass.) of the coomassie-blue stainedSDS-PAGE gel.

EXAMPLE 5 Cloning and Expression of Dac g V

Dactylis glomerata pollen was purchased from Greer Laboratories (Lenoir,N.C.). RNA was isolated as previously described in PCT/US92/05661(WO93/01213) and polyA+ RNA was isolated using MICRO-FAST TRACK® mRNAisolation kit from Invitrogen (San Diego, Calif.). Double stranded cDNAwas made with the BRL cDNA SYNTHESIS PLUS® kit (Gaithersburg, Md.). AcDNA library was made in lgt10 using the cDNA CLONING SYSTEM-lGT10®(Amersham, Arlington Heights, Ill.). The D. glomerata double strandedcDNA was ligated with adaptor arms, containing Eco RI, Bam HI, Kpn I andNco I restriction sites and ligated into (lambda) gt10 vector arms usingthe manufacturer's suggested protocols. The library was packaged andtitred also using the manufacturer's suggested protocols. The librarywas plated out and over 100,000 independent phage plaques were screenedusing random primed (RANDOM PRIMED DNA LABELING KIT®, BoehrningerMannheim Corporation, Indianopolis, Ind.) or nick-translated probe[Sambrook J et al. Molecular Cloning: A Laboratory Manual. Cold SpringHarbor: Cold Spring Harbor Laboratory, 1989]. The library was screenedwith the 1.2 kb Lol p V clone 12R cDNA [Singh MB et al, Proc Natl AcadSci USA, 1991; 88: 1384–1388].

There were many positive clones identified in the first screen. Severalclones were picked using standard techniques [Sambrook J et al.Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold SpringHarbor Laboratory, 1989] and dilutions of high-titred phage stocks werere-screened using the same Lol p V clone 12R probe. The phage stockswere prepared using standard techniques [Sambrook J et al. MolecularCloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring HarborLaboratory, 1989]. Positive clones were again picked, high-titred stocksprepared as before and serial dilutions were prepared for tertiaryscreening with the Lol p V clone 12R probe. Six phage clones, 228, 235,236, 259, 267, and 285, were positive after this tertiary screening andhigh titred stocks were prepared as described [Sambrook J et al.Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold SpringHarbor Laboratory, 1989]. The cDNA inserts were isolated from theselected phage using standard techniques [Sambrook J et al. MolecularCloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring HarborLaboratory, 1989]. The insert from clone 228 was approximately 500 basepairs (bp). The insert from clone 235 was approximately 1,000 bp. Theinsert from clone 236 was approximately 1200 bp. The insert from clone259 was approximately 1,200 bp. The insert from clone 267 wasapproximately 1,000 bp. The insert from clone 285 was approximately 800bp. The isolated inserts were cloned into appropriately digested pUC18and/or pUC19 for subsequent analysis. The cDNA inserts were sequencedusing the SEQUENASE® kits (USB, Cleveland, Ohio) based on the standarddideoxy chain termination method of Sanger et al. [Sanger F et al. ProcNatl Acad Sci USA, 1977; 74: 5460–5463].

Partial sequences for all of the clones were determined. All were foundto contain Dac g V sequences by comparison with Lol p V clone 12Rsequence (SEQ ID NO:2) [Ong EK et al. Gene, 1993; 134: 235–240]. Thepartial translated sequences of clones 235 and 236 were very similar toeach other, although they started at different sites in the sequence(not shown), and appear to represent one isoform of Dac g V. The partialtranslated sequence of clone 259 was different from that of clones 235and 236 and appear to represent a second isoform of Dac g V. The partialtranslated sequence of clone 259 is most homologous to the sequence ofLol p V clone 12R (SEQ ID NO:2) [Ong EK et al. Gene 1993; 134: 235–240].The partial translated sequences of clones 235 and 236 are most closelyhomologous to the sequence of Lol p V clone 19R [Ong EK et al. Gene1993; 134: 235–240].

Clone 259 was sequenced in its entirety. It was sequenced from both endsusing standard forward and reverse primers (New England Biolabs,Beverly, Mass.). Subconstructs were prepared by digestion of isolatedinsert with Eco RI and Pst I and the fragments were cloned intoappropriately digested pUC18 for internal sequencing. The Eco RI/Pst Iinsert that corresponded to the 5′ portion of the Dac g V gene wasisolated and further digested with Stu I or Sau 3A and Xho I and ligatedinto appropriately digested pUC19 for further sequence analysis. Thenucleotide (SEQ ID NO:57) and deduced amino acid (SEQ ID NO:58) sequenceof clone 259 is shown in FIG. 16. Nucleotides 1–25 correspond to adaptorsequence. The sequence ends with the poly A tract; the adaptor sequenceis not shown at the 3′ end of the sequence. The nucleotide sequence from700 to 1181 is only preliminary and some bases may be misidentified. Forexample, nucleotide 712 has been tentatively identified as a “C”.However, this is the third position of the codon encoding Gly 196 andthe presence of another nucleotide at residue 712 would not change thepredicted amino acid. It is difficult to sequence the Group V grassallergens due to their high GC content.

Clone 236 and 259 have been deposited with the ATCC.

As Group V allergens tend to have very conserved regions, the major Tcell epitope containing peptides of Lol p V as described herein, arelikely to be the major T cell epitopes of Dac g V, particularly wherethe regions are highly conserved between the related grasses.

1. A composition consisting of a combination of peptides selected fromthe group of combinations consisting of: COMBINATION (a): LPIX-4 (SEQ IDNO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-16 (SEQ IDNO:18), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ ID NO:22); COMBINATION(b): LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22); LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ IDNO:22); COMBINATION (c): LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7),LPIX-6 (SEQ ID NO:8), and LPIX-20 (SEQ ID NO:22); COMBINATION (d):LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8),LPIX-11 (SEQ ID NO: 13), LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18),LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ ID NO:22); COMBINATION (e):LPIX-4 (SEQ ID NO:6), LPIX-5 (SEQ ID NO:7), LPIX-6 (SEQ ID NO:8), LPIX-8(SEQ ID NO: 10), LPIX-9 (SEQ ID NO: 11), LPIX-11 (SEQ ID NO: 13),LPIX-12 (SEQ ID NO:14), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),LPIX-19 (SEQ ID NO: 21), LPIX-20 (SEQ ID NO:22), LPIX-23 (SEQ ID NO:25),and LPIX-26 (SEQ ID NO:28); COMBINATION (f): LPIX-4 (SEQ ID NO:6),LPIX-11 (SEQ ID NO: 13), LPIX-16 (SEQ ID NO:18), and LPIX-20 (SEQ IDNO:22); COMBINATION (g): LPIX-4 (SEQ ID NO:6), LPIX-11 (SEQ ID NO: 13),LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ ID NO:22); LPIX-4 (SEQ IDNO:6), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQID NO:22); COMBINATION (h): LPIX-5 (SEQ ID NO:7), LPIX-11 (SEQ ID NO:13), LPIX-16 (SEQ ID NO:18), and LPIX-20 (SEQ ID NO:22); COMBINATION(i): LPIX-5 (SEQ ID NO:7), LPIX-11 (SEQ ID NO: 13), LPIX-17 (SEQ IDNO:19), and LPIX-20 (SEQ ID NO:22); COMBINATION (j): LPIX-5 (SEQ IDNO:7), LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQID NO:22); COMBINATION (k): LPIX-11 (SEQ ID NO: 13), LPIX-16 (SEQ IDNO:18), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ ID NO:22); LPIX-4 (SEQID NO:6), LPIX-11 (SEQ ID NO: 13), and LPIX-20 (SEQ ID NO:22);COMBINATION (l): LPIX-4 (SEQ ID NO:6), LPIX-16 (SEQ ID NO:18), andLPIX-20 (SEQ ID NO:22); COMBINATION (m): LPIX-4 (SEQ ID NO:6), LPIX-17(SEQ ID NO:19), and LPIX-20 (SEQ ID NO:22); COMBINATION (n): LPIX-5 (SEQID NO:7), LPIX-11 (SEQ ID NO: 13), and LPIX-20 (SEQ ID NO:22);COMBINATION (o): LPIX-5 (SEQ ID NO:7), LPIX-16(SEQ ID NO:18), andLPIX-20 (SEQ ID NO:22); COMBINATION (p): LPIX-11 (SEQ ID NO: 13),LPIX-16 (SEQ ID NO:18), and LPIX-20 (SEQ ID NO:22); COMBINATION (q):LPIX-11 (SEQ ID NO: 13), LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ IDNO:22); COMBINATION (r): LPIX-16 (SEQ ID NO:18), LPIX-17 (SEQ ID NO:19),and LPIX-20 (SEQ ID NO:22); COMBINATION (s): LPIX-5 (SEQ ID NO:7),LPIX-17 (SEQ ID NO:19), and LPIX-20 (SEQ ID NO:22); COMBINATION (t):LPIX-4 (SEQ ID NO:6), LPIX-20 (SEQ ID NO:22); COMBINATION (u): LPIX-5(SEQ ID NO:7), and LPIX-20 (SEQ ID NO:22); COMBINATION (v): LPIX-6 (SEQID NO:8), and LPIX-20 (SEQ ID NO:22); COMBINATION (w): LPIX-11 (SEQ IDNO: 13), and LPIX-20 (SEQ ID NO:22); COMBINATION (x): LPIX-12 (SEQ IDNO:14), and LPIX-20 (SEQ ID NO:22); COMBINATION (y): LPIX-16 (SEQ IDNO:18), and LPIX-20 (SEQ ID NO:22); and COMBINATION (z): LPIX-17 (SEQ IDNO:19), and LPIX-20 (SEQ ID NO:22).
 2. A composition consisting of thecomposition of claim 1 and a pharmaceutically acceptable carrier ordiluent.